A Malpighian Tubule-Specific P450 Gene SlCYP9A75a Contributes to Xenobiotic Tolerance in Spodoptera litura.

Phytophagous insects are more predisposed to evolve insecticide resistance than other insect species due to the "preadaptation hypothesis". Cytochrome P450 monooxygenases have been strongly implicated in insecticide and phytochemical detoxification in insects. In this study, RNA-seq results reveal that P450s of Spodoptera litura, especially the CYP3 clan, are dominant in cyantraniliprole, nicotine, and gossypol detoxification. The expression of a Malpighian tubule-specific P450 gene, SlCYP9A75a, is significantly upregulated in xenobiotic treatments except α-cypermethrin. The gain-of-function and loss-of-function analyses indicate that SlCYP9A75a contributes to cyantraniliprole, nicotine, and α-cypermethrin tolerance, and SlCYP9A75a is capable of binding to these xenobiotics. This study indicates the roles of inducible SlCYP9A75a in detoxifying man-made insecticides and phytochemicals and may provide an insight into the development of cross-tolerance in omnivorous insects.

A sperm-activating trypsin-like protease from the male reproductive tract of Spodoptera litura: Proteomic identification, sequence characterization, gene expression profile, RNAi and the effects of ionizing radiation.

Like other lepidopteran insects, males of the tobacco cutworm moth, Spodoptera litura produce two kinds of spermatozoa, eupyrene (nucleate) and apyrene (anucleate) sperm. Formed in the testis, both kinds of sperm are released into the male reproductive tract in an immature form and are stored in the duplex region of the tract. Neither type of sperm is motile at this stage. When stored apyrene sperm from the duplex are treated in vitro with an extract of the prostatic region of the male tract, or with mammalian trypsin, they become motile; activation is greater and achieved more rapidly with increasing concentration of extract or enzyme. The activating effect of prostatic extract is blocked by soybean trypsin inhibitor (SBTI), also in a dose-dependent way. These results suggest that the normal sperm-activating process is due to an endogenous trypsin-like protease produced in the prostatic region. Proteomic analysis of S. litura prostatic extracts revealed a Trypsin-Like Serine Protease, TLSP, molecular weight 27 kDa, whose 199-residue amino acid sequence is identical to that of a predicted protein from the S. litura genome and is highly similar to predicted proteins encoded by genes in the genomes of several other noctuid moth species. Surprisingly, TLSP is only distantly related to Serine Protease 2 (initiatorin) of the silkmoth, Bombyx mori, the only identified lepidopteran protein so far shown to activate sperm. TLSP has features typical of secreted proteins, probably being synthesized as an inactive precursor zymogen, which is later activated by proteolytic cleavage. cDNA was synthesized from total RNA extracted from the prostatic region and was used to examine TLSP expression using qPCR. tlsp mRNA was expressed in both the prostatic region and the accessory glands of the male tract. Injection of TLSP-specific dsRNA into adult males caused a significant reduction after 24 h in tlsp mRNA levels in both locations. The number of eggs laid by females mated to adult males that were given TLSP dsRNA in 10 % honey solution, and the fertility (% hatched) of the eggs were reduced. Injecting pupae with TLSP dsRNA caused the later activation of apyrene sperm motility by adult male prostatic extracts to be significantly reduced compared to controls. Exposure of S. litura pupae to ionizing radiation significantly reduced expression of tlsp mRNA in the prostatic part and accessory gland of irradiated males in both the irradiated generation and also in their (unirradiated) F1 progeny. The implications of these findings for the use of the inherited sterility technique for the control of S. litura and other pest Lepidoptera are discussed.

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.

Insecticide resistance in Australian Spodoptera frugiperda (J.E. Smith) and development of testing procedures for resistance surveillance.

Spodoptera frugiperda (J.E. Smith) is a highly invasive noctuid pest first reported in northern Australia during early 2020. To document current status of resistance in S. frugiperda in Australia, insecticide toxicity was tested in field populations collected during the first year of establishment, between March 2020 and March 2021. Dose-response was measured by larval bioassay in 11 populations of S. frugiperda and a susceptible laboratory strain of Helicoverpa armigera. Emamectin benzoate was the most efficacious insecticide (LC50 0.023µg/ml) followed by chlorantraniliprole (LC50 0.055µg/ml), spinetoram (LC50 0.098µg/ml), spinosad (LC50 0.526µg/ml), and methoxyfenozide (1.413µg/ml). Indoxacarb was the least toxic selective insecticide on S. frugiperda (LC50 3.789µg/ml). Emamectin benzoate, chlorantraniliprole and methoxyfenozide were 2- to 7-fold less toxic on S. frugiperda compared with H. armigera while spinosyns were equally toxic on both species. Indoxacarb was 28-fold less toxic on S. frugiperda compared with H. armigera. There was decreased sensitivity to Group 1 insecticides and synthetic pyrethroids in S. frugiperda compared with H. armigera: toxicity was reduced up to 11-fold for methomyl, 56 to 199-fold for cyhalothrin, and 44 to 132-fold for alpha cypermethrin. Synergism bioassays with metabolic inhibitors suggest involvement of mixed function oxidase in pyrethroid resistance. Recommended diagnostic doses for emamectin benzoate, chlorantraniliprole, spinetoram, spinosad, methoxyfenozide and indoxacarb are 0.19, 1.0, 0.75, 6, 12 and 48µg/µl, respectively.

A diagnostic LAMP assay for rapid identification of an invasive plant pest, fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae).

Fall armyworm (FAW), Spodoptera frugiperda (Lepidoptera: Noctuidae), is a highly polyphagous invasive plant pest that has expanded its global geographic distribution, including recently into much of Australia. Rapid diagnostic tests are required for identification of FAW to assist subsequent management and control. We developed a new loop-mediated isothermal amplification (LAMP) assay based on the mitochondrial cytochrome c oxidase subunit I (COI) gene for accurate and timely diagnosis of FAW in the field. The specificity of the new assay was tested against a broad panel of twenty non-target noctuids, including eight other Spodoptera species. Only S. frugiperda samples produced amplification within 20 min, with an anneal derivative temperature of 78.3 ± 0.3 °C. A gBlock dsDNA fragment was developed and trialled as a synthetic positive control, with a different anneal derivative of 81 °C. The new FAW LAMP assay was able to detect FAW DNA down to 2.4 pg, similar to an existing laboratory-based real-time PCR assay. We also trialled the new FAW assay with a colorimetric master mix and found it could successfully amplify positive FAW samples in half the time compared to an existing FAW colorimetric LAMP assay. Given the high sensitivity and rapid amplification time, we recommend the use of this newly developed FAW LAMP assay in a portable real-time fluorometer for in-field diagnosis of FAW.

The fitness advantages of bistrifluron resistance related to chitin synthase A in Spodoptera litura (Fab.) (Noctuidae: Lepidoptera).

BACKGROUND: Spodoptera litura is one of the major agricultural pests in China, and it has developed serious resistance to many traditional chemical insecticides. In the present study, the bistrifluron-resistant (Bis-SEL) strain accompanied by a higher oviposition, 113.8-fold RR compared to the bistrifluron-susceptible (Bis-UNSEL) strain, was obtained by bidirectional screening. A comparison of their gonad coefficiency and genes related to oviposition or resistance was used to elucidate the resurgence mechanism. RESULTS: The ovarian index, oviposition, and potential egg production in the Bis-SEL strain of female adults were significantly higher than those in the Bis-UNSEL strain, and the length of ovariole in the Bis-SEL strain was also significantly elongated. The protein contents of vitellogenin (Vg) and vitellogenin receptor (VgR) in the Bis-UNSEL strain were lower than those in the Bis-SEL strain, consistent with their gene expressions levels, and there was a significantly positive linear correlation between Vg and VgR protein contents, further confirming that resistant strains have high reproductive fitness. Moreover, the chitin synthase A in the Bis-SEL strain was clearly up-regulated, and a mutation (H866Y) near the QRRRW in the catalytic domain caused a rise in the hydrogen bond between UDP-GlcNAc and chitin synthase, and its chitin content was higher than that in the Bis-UNSEL strain. Nevertheless, the sensitivity of the Bis-SEL strain to bistrifluron was significantly recovered when it was knocked down though RNA interference. CONCLUSION: The fitness advantages of bistrifluron resistance may be related to the up-regulation and mution of chitin synthase A. © 2021 Society of Chemical Industry.

The prostanoids, thromboxanes, mediate hemocytic immunity to bacterial infection in the lepidopteran Spodoptera exigua.

We report on a new insect prostanoid in a lepidopteran insect, Spodoptera exigua. Thromboxane B2 (TXB2) was detected by LC-MS/MS in extracts of larval epidermis, midgut, fat body and hemocytes, with highest amounts in hemocytes (about 300 ng/g tissue with substantial variation). Thromboxane A2 (TXA2) is an unstable intermediate that is non-enzymatically hydrolyzed into the stable TXB2. In S. exigua, both thromboxanes mediate at least two cellular immune responses to bacterial infection, hemocyte-spreading behavior and nodule formation. At the molecular level, a TXA2 synthase (SeTXAS) was identified from a group of 139 S. exigua cytochrome P450 monooxygenases. SeTXAS was highly similar to mammalian TXAS genes and is expressed in all developmental stages and four tested larval tissues. Immune challenge significantly enhanced SeTXAS expression, especially in hemocytes. RNA interference (RNAi) injections using gene-specific double stranded RNA led to reduced SeTXAS expression and suppressed the cellular immune responses, which were rescued following TXA2 or TXB2 injections. Unlike other PGs, TXA2 or TXB2 did not influence oocyte development in adult females. We infer that thromboxanes are present in insect tissues, where they mediate innate immune responses.

Homodimerization of a glycoside hydrolase family GH1 ß-glucosidase suggests distinct activity of enzyme different states.

In this work, we investigated how activity and oligomeric state are related in a purified GH1 ß-glucosidase from Spodoptera frugiperda (Sfßgly). Gel filtration chromatography coupled to a multiple angle light scattering detector allowed separation of the homodimer and monomer states and determination of the dimer dissociation constant (KD ), which was in the micromolar range. Enzyme kinetic parameters showed that the dimer is on average 2.5-fold more active. Later, we evaluated the kinetics of homodimerization, scanning the changes in the Sfßgly intrinsic fluorescence over time when the dimer dissociates into the monomer after a large dilution. We described how the rate constant of monomerization (koff ) is affected by temperature, revealing the enthalpic and entropic contributions to the process. We also evaluated how the rate constant (kobs ) by which equilibrium is reached after dimer dilution behaves when varying the initial Sfßgly concentration. These data indicated that Sfßgly dimerizes through the conformational selection mechanism, in which the monomer undergoes a conformational exchange and then binds to a similar monomer, forming a more active homodimer. Finally, we noted that conformational selection reports and experiments usually rely on a ligand whose concentration is in excess, but for homodimerization, this approach does not hold. Hence, since our approach overcomes this limitation, this study not only is a new contribution to the comprehension of GH1 ß-glucosidases, but it can also help to elucidate protein interaction pathways.

Predator-induced stress influences fall armyworm immune response to inoculating bacteria.

The insect innateimmunesystem is assorted into two general categories, cellular and humoral immunity. Aside from direct challenge by invaders, predation risk can be perceived as odors, sounds or nearness. In this study, we evaluated influence of predation risk by the predatory bug Podisus maculiventris on immunity of an herbivore Spodoptera frugiperda. Under the predator-induced stress combined with Escherichia coli inoculation, several larval physiological parameters of S. frugiperda were studied, including body mass, nodulation, and phenoloxidase activity. Our findings offernew evidence that provides insight into the immunological mechanism of predator-induced stress effects on prey species.

Transcriptomic analysis of the testicular fusion in Spodoptera litura.

BACKGROUND: Lepidoptera is one group of the largest plant-feeding insects and Spodoptera litura (Lepidoptera: Noctuidae) is one of the most serious agricultural pests in Asia countries. An interesting and unique phenomenon for gonad development of Lepidoptera is the testicular fusion. Two separated testes fused into a single one during the larva-to-pupa metamorphosis, which is believed to contribute to sperm production and the prevalence in field. To study the molecular mechanism of the testicular fusion, RNA sequencing (RNA-seq) experiments of the testes from 4-day-old sixth instar larvae (L6D4) (before fusion), 6-day-old sixth instar larvae (L6D6, prepupae) (on fusing) and 4-day-old pupae (P4D) (after fusion) of S. litura were performed. RESULTS: RNA-seq data of the testes showed that totally 12,339 transcripts were expressed at L6D4, L6D6 and P4D stages. A large number of differentially expressed genes (DEGs) were up-regulated from L6D4 to L6D6, and then more genes were down-regulated from L6D6 to P4D. The DEGs mainly belongs to the genes related to the 20E signal transduction pathway, transcription factors, chitin metabolism related enzymes, the families of cytoskeleton proteins, extracellular matrix (ECM) components, ECM-related protein, its receptor integrins and ECM-remodeling enzymes. The expression levels of these genes that were up-regulated significantly during the testicular fusion were verified by qRT-PCR. The matrix metalloproteinases (MMPs) were found to be the main enzymes related to the ECM degradation and contribute to the testicular fusion. The testis was not able to fuse if MMPs inhibitor GM6001 was injected into the 5th abdomen region at L6D6 early stage. CONCLUSIONS: The transcriptome and DEGs analysis of the testes at L6D4, L6D6 and P4D stages provided genes expression information related to the testicular fusion in S. litura. These results indicated that cytoskeleton proteins, ECM-integrin interaction genes and ECM-related proteins were involved in cell migration, adhesion and fusion during the testicular fusion. The ECM degradation enzymes MMPs probably play a critical role in the fusion of testis.

Initial detections and spread of invasive Spodoptera frugiperda in China and comparisons with other noctuid larvae in cornfields using molecular techniques.

The fall armyworm, Spodoptera frugiperda, is a species native to the Americas and has spread to many countries in Africa and Asia in recent years. Proactive actions for potential invasion of S. frugiperda to China coordinated by government agencies and agricultural extension systems resulted in timely detection in January 2019 in Yunnan province neighboring onto Myanmar. The extensive monitoring in southern provinces of China since February 2019 resulted in dynamic tracking of S. frugiperda spreading to 13 provincial regions in China within 4 months by May 10, 2019, which is crucial for timely management actions in the fields. The first detections of S. frugiperda (corn strain) in China were confirmed using cytochrome oxidase subunit 1 (CO1) and triosephosphate isomerase (Tpi) genes molecular marker method. In addition to S. frugiperda, larvae of three other noctuid species with similar morphological appearance (S. litura, S. exigua and Mythimna separata) can occur simultaneously and cause similar damage in cornfields in southern China. Thus, we can use both morphological and molecular marker methods to compare larval stages of four noctuid species. Further, we discuss the risk of potential spread of invasive S. frugiperda to other regions and impact on corn production in China.

Functional interaction of bacterial virulence factors of Xenorhabdus nematophila with a calcium-independent cytosolic PLA2 of Spodoptera exigua.

Phospholipase A2 (PLA2) hydrolyzes the ester bond of phospholipids (PLs) at sn-2 and releases free fatty acids and lysophospholipids that are subsequently changed into various signal molecules to mediate various physiological processes. Numerous PLA2s are known in various biological systems and can be divided into at least 16 groups. Although different PLA2s recently have been annotated from several insect species, physiological roles are known for only a few genes. Two calcium-independent PLA2s (Se-iPLA2A and Se-iPLA2B) are known in the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae). We generated and purified a recombinant Se-iPLA2B (rSe-iPLA2B) using a bacterial expression system and analyzed the enzyme kinetics. rSe-iPLA2B exhibited catalytic activities against both arachidonyl (AA)-PL and non-AA-PL substrates. It was highly susceptible to iPLA2-specific inhibitor, but insensitive to inhibitors specific to secretory PLA2s or calcium-dependent cytosolic PLA2s. Increasing calcium concentrations prevented enzyme activity, and culture medium of an entomopathogenic bacterium, Xenorhabdus nematophila, or its organic extracts significantly inhibited enzyme activity. Binding assays of rSe-iPLA2B with known secondary metabolites identified from X. nematophila indicated that benzylideneacetone was the most potent inhibitor with a high binding affinity at 0.2 µM against rSe-iPLA2B. Furthermore, rSe-iPLA2B catalyzed the release of fatty acids from PLs extracted from S. exigua fat body, suggesting its physiological role in maintaining PL integrity. All these catalytic activities indicate that Se-iPLA2B has the typical biochemical properties of other iPLA2s. Its high binding affinity to secondary metabolites of X. nematophila suggests that it is a molecular target of X. nematophila, an entomopathogen.

Activation of CncC pathway by ROS burst regulates cytochrome P450 CYP6AB12 responsible for λ-cyhalothrin tolerance in Spodoptera litura.

Frequent insecticide use poses an environmental hazard and also selects for insecticide tolerance. Increased metabolic detoxification by cytochrome P450 monooxygenases (P450s) is the most common mechanism of insecticide tolerance. However, the underlying regulatory mechanisms remain unknown. We studied the midgut-specific P450 gene, CYP6AB12, associated with λ-cyhalothrin tolerance. Its regulatory pathway was investigated in the tobacco cutworm, Spodoptera litura (Fabricius). P450 activities and CYP6AB12 transcript levels increased after λ-cyhalothrin exposure. Inhibiting P450 activities with piperonyl butoxide and silencing CYP6AB12 by double-stranded RNA (dsRNA) injection decreased larval tolerance to λ-cyhalothrin. λ-Cyhalothrin exposure induced the expression of the cap 'n' collar isoform C (CncC) and muscle aponeurosis fibromatosis (Maf), increased hydrogen peroxide (H2O2) contents and elevated antioxidant enzyme activities. CncC knockdown by dsRNA feeding suppressed CYP6AB12 expression and decreased larval tolerance to λ-cyhalothrin. In contrast, application of the CncC agonist curcumin induced CYP6AB12 expression and enhanced insecticide tolerance. Ingestion of the reactive oxygen species (ROS) scavenger N-acetylcysteine reduced H2O2 accumulation, suppressed the expression of CncC, Maf and CYP6AB12 and led to increased larval susceptibility to λ-cyhalothrin. The results demonstrate that in S. litura, λ-cyhalothrin induces cytochrome P450 CYP6AB12 via elicitation of the ROS burst and activation of the CncC pathway.

Identification of four caspase genes from Spodoptera exigua (Lepidoptera: Noctuidae) and their regulations toward different apoptotic stimulations.

Apoptosis plays critical roles in multiple biological processes in multicellular organisms. Caspases are known as important participators and regulators of apoptosis. Here, four novel caspase genes of Spodoptera exigua were cloned and characterized, which were designated as SeCasp-1, SeCasp-6, SeCasp-7 and SeCasp-8. Analysis of the putative encoded protein sequences of these SeCasps indicated that SeCasp-1 and SeCasp-7 were possible homologs of executor caspases; SeCasp-8 was a possible homolog of initiator caspases; and SeCasp-6 was a unique caspase of S. exigua that shares low similarity with all the identified insect caspases. Based on baculovirus expression system analyses, SeCasp-1 exhibited similar caspase activity to human caspase-1, -3, -4, -6, -8 and -9; SeCasp-6 presented similar caspase activity to human caspase-2, -3, -4, -6, -8 and -9; SeCasp-7 exhibited similar caspase activity to human caspase-2, -3 and -6; and SeCasp-8 presented similar caspase activity only to human caspase-8. Induction with different chemicals revealed that SeCasp-1 showed extreme upregulation after 24 h in the treated fat body cell line (IOZCAS-Spex-II) of S. exigua. Developmental expression analysis revealed that SeCasp-1 was highly transcribed in the larval stages, while SeCasp-6, SeCasp-7, SeCasp-8 were down-regulated. The in vivo detection of the relative expression levels of SeCasps in S. eixgua larvae inoculated with different pathogens suggested that SeCasp-1 was sensitive to Bacillus thuringiensis infection and that SeCasp-6 was sensitive to baculovirus infection. SeCasp-7 and SeCasp-8 showed slight changes under either in vitro chemical apoptosis induction or in vivo pathogen infection.

Synthesis and biological activities of two camptothecin derivatives against Spodoptera exigua.

Camptothecin (CPT), a natural alkaloid isolated from Camptotheca acuminata Decne, is found to show potential insecticidal activities with unique action mechanisms by targeting at DNA-topoisomease I (Top1) complex and inducing cell apoptosis. To improve the efficacy against insect pests, two camptothecin (CPT) derivatives were synthesized through introducing two functional groups, 2-nitroaminoimidazoline and 1-chloro-2-isocyanatoethane by esterification reaction. The insecticidal activities of these two derivatives were evaluated at contact toxicity, cytotoxicity and topoisomerase I (Top1) inhibitory activities comparing with CPT and hydroxyl-camptothecin (HCPT). Results showed that compound a, synthesized by introducing 2-nitroaminoimidazoline to CPT, apparently increased contact toxicity to the third larvae of beet armyworm, Spodoptera exigua, and cytotoxicity to IOZCAS-Spex-II cells isolated from S. exigua. However, the inhibition on DNA relaxation activity of Top1 was reduced to less than 5 percentage even at high concentrations (50 and 100 µM). For introducing 1-chloro-2-isocyanatoethane to HCPT, the contact toxicity, cytotoxicity and Top1 inhibitory activity of synthesized compound b were increased significantly compared to CPT and HCPT. These results suggested that both synthesized compounds possessed high efficacy against S. exigua by targeting at Top1 (compound b) or novel mechanism of action (compound a).

Asatone and Isoasatone A Against Spodoptera litura Fab. by Acting on Cytochrome P450 Monoxygenases and Glutathione Transferases.

Asatone and isoasatone A from Asarum ichangense Cheng were determined to be defensive compounds to some insects in a previous investigation. However, the anti-insect activity mechanisms to caterpillar are still unclear. The compounds asatone and isoasatone A from A. ichangense were induced by Spodoptera litura. The anti-insect activity of asatone and isoasatone A to S. litura was further tested by weight growth rate of the insect through a diet experiment. Isoasatone A showed a more significant inhibitory effect on S. litura than asatone on the second day. The concentration of asatone was higher than isoasatone A in the second instar larvae of S. litura after 12 h on the feeding test diet. Both compounds caused mid-gut structural deformation and tissue decay as determined by mid-gut histopathology of S. litura. Furthermore, some detoxification enzyme activity were measured by relative expression levels of genes using a qPCR detecting system. Asatone inhibited the gene expression of the cytochrome P450 monooxygenases (P450s) CYP6AB14. Isoasatone A inhibited the relative expression levels of CYP321B1, CYP321A7, CYP6B47, CYP6AB14, and CYP9A39. Asatone increased the relative gene expression of the glutathione transferases (GSTs) SIGSTe1 and SIGSTo1, in contrast, isoasatone A decreased the relative gene expression of SIGSTe1 by about 33 fold. Neither compound showed an effect on acetylcholinesterase SIAce1 and SIAce2. The mechanism of anti-insect activity by both compounds could be explained by the inhibition of enzymes P450s and GSTs. The results provide new insights into the function of unique secondary metabolites asatone and isoasatone A in genus Asarum, and a new understanding of why A. ichangense is largely free of insect pests.

Synthesis, Biological Evaluation and In Silico Computational Studies of 7-Chloro-4-(1H-1,2,3-triazol-1-yl)quinoline Derivatives: Search for New Controlling Agents against Spodoptera frugiperda (Lepidoptera: Noctuidae) Larvae.

The insecticidal and antifeedant activities of five 7-chloro-4-(1H-1,2,3-triazol-1-yl)quinoline derivatives were evaluated against the maize armyworm, Spodoptera frugiperda (J.E. Smith). These hybrids were prepared through a copper-catalyzed azide alkyne cycloaddition (CuAAC, known as a click reaction) and displayed larvicidal properties with LD50 values below 3 mg/g insect, and triazolyl-quinoline hybrid 6 showed an LD50 of 0.65 mg/g insect, making it 2-fold less potent than methomyl, which was used as a reference insecticide (LD50 = 0.34 mg/g insect). Compound 4 was the most active antifeedant derivative (CE50 = 162.1 µg/mL) with a good antifeedant index (56-79%) at concentrations of 250-1000 µg/mL. Additionally, triazolyl-quinoline hybrids 4-8 exhibited weak inhibitory activity against commercial acetylcholinesterase from Electrophorus electricus (electric-eel AChE) (IC50 = 27.7 µg/mL) as well as low anti-ChE activity on S. frugiperda larvae homogenate (IC50 = 68.4 µg/mL). Finally, molecular docking simulations suggested that hybrid 7 binds to the catalytic active site (CAS) of this enzyme and around the rim of the enzyme cavity, acting as a mixed (competitive and noncompetitive) inhibitor like methomyl. Triazolyl-quinolines 4-6 and 8 inhibit AChE by binding over the perimeter of the enzyme cavity, functioning as noncompetitive inhibitors. The results described in this work can help to identify lead triazole structures from click chemistry for the development of insecticide and deterrent products against S. frugiperda and related insect pests.

Overexpression of PGE2 synthase by in vivo transient expression enhances immunocompetency along with fitness cost in a lepidopteran insect.

Prostaglandins (PGs) mediate various physiological functions in insects. Specifically, PGE2 is known to mediate immunity and egg-laying behavior in the beet armyworm, Spodoptera exigua A PGE2 synthase 2 (Se-PGES2) has been identified to catalyze the final step to produce PGE2 in S. exigua Its expression is inducible in response to immune challenge. Inhibition of the gene expression results in immunosuppression. In contrast, any physiological alteration induced by its uncontrolled overexpression was not recognized in insects. This study used the in vivo transient expression (IVTE) technique to induce overexpression and assessed subsequent physiological alteration in S. exiguaSe-PGES2 was cloned into a eukaryotic expression vector and transfected to Sf9 cells to monitor its heterologous expression. The Sf9 cells expressed the recombinant Se-PGES2 (rSe-PGES2) at an expected size (∼47 kDa), which was localized in the cytoplasm. The recombinant expression vector was then used to transfect larvae of S. exigua Hemocytes collected from the larvae treated with IVTE expressed the rSe-PGES2 gene for at least 48 h. The larvae treated with IVTE exhibited an enhanced competency in cellular immune response measured by hemocyte nodule formation. In addition, IVTE treatment of Se-PGES2 induced gene expression of antimicrobial peptides without any immune challenge. The larvae treated with IVTE became significantly resistant to infection of an entomopathogenic nematode, Steinernema monticolum, or to infection to its symbiotic bacterium, Xenorhabdus hominickii However, IVTE-treated S. exigua larvae suffered from reduced pupal size and fecundity.

Characterization of the 20S proteasome of the lepidopteran, Spodoptera frugiperda.

Multiple complexes of 20S proteasomes with accessory factors play an essential role in proteolysis in eukaryotic cells. In this report, several forms of 20S proteasomes from extracts of Spodoptera frugiperda (Sf9) cells were separated using electrophoresis in a native polyacrylamide gel and examined for proteolytic activity in the gel and by Western blotting. Distinct proteasome bands isolated from the gel were subjected to liquid chromatography-tandem mass spectrometry and identified as free core particles (CP) and complexes of CP with one or two dimers of assembly chaperones PAC1-PAC2 and activators PA28γ or PA200. In contrast to the activators PA28γ and PA200 that regulate the access of protein substrates to the internal proteolytic chamber of CP in an ATP-independent manner, the 19S regulatory particle (RP) in 26S proteasomes performs stepwise substrate unfolding and opens the chamber gate in an ATP-dependent manner. Electron microscopic analysis suggested that spontaneous dissociation of RP in isolated 26S proteasomes leaves CPs with different gate sizes related presumably to different stages in the gate opening. The primary structure of 20S proteasome subunits in Sf9 cells was determined by a search of databases and by sequencing. The protein sequences were confirmed by mass spectrometry and verified by 2D gel electrophoresis. The relative rates of sequence divergence in the evolution of 20S proteasome subunits, the assembly chaperones and activators were determined by using bioinformatics. The data confirmed the conservation of regular CP subunits and PA28γ, a more accelerated evolution of PAC2 and PA200, and especially high divergence rates of PAC1.

Toll/IMD signal pathways mediate cellular immune responses via induction of intracellular PLA 2 expression.

Phospholipase A2 (PLA2 ) hydrolyzes fatty acids from phospholipids at the sn-2 position. Two intracellular PLA2 s, iPLA2 A and iPLA2 B, have been found in Spodoptera exigua. Both are calcium-independent cellular PLA2 . Their orthologs have been found in other insects. These two iPLA2 s are different in ankyrin motif of N terminal region. The objective of this study was to determine whether Toll/immune deficiency (IMD) signal pathways could mediate cellular immune responses via induction of iPLA2 expression. Both iPLA 2 s were expressed in all developmental stages of S. exigua, showing the highest expression in the adult stage. During larval stage, hemocyte is the main tissue showing expression of these iPLA2 s. Both iPLA2 s exhibited similar expression patterns after immune challenge with different microbial pathogens such as virus, bacteria, and fungi. Promoter component analysis of orthologs encoded in S. frugiperda indicated nuclear factor-κB- and Relish-responsible elements on their promoters, suggesting their expression in S. exigua under Toll/IMD immune signaling pathways. RNA interference (RNAi) of MyD88 or Pelle under Toll pathway suppressed inducible expression levels of both iPLA2 s in response to Gram-positive bacteria containing Lys-type peptidoglycan or fungal infection. In contrast, RNAi against Relish under IMD pathway suppressed both iPLA2 s in response to infection with Gram-negative bacteria. Under RNAi conditions, hemocytes significantly lost cellular immune response measured by nodule formation. However, addition of arachidonic acid (a catalytic product of PLA2 ) rescued such immunosuppression. These results suggest that Toll/IMD signal pathways can mediate cellular immune responses via eicosanoid signaling by inducing iPLA2 expression.