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PubMed1996

A common pathway for p10 and calyx proteins in progressive stages of polyhedron envelope assembly in AcMNPV-infected Spodoptera frugiperda larvae.

The assembly of the polyhedron envelope in baculovirus-infected cells has been the subject of several studies, yet it is still poorly understood. We have used immunogold-labelled antibodies to two baculovirus proteins, p10 and calyx (also referred to as polyhedron envelope protein or PEP), to follow envelope assembly in AcMNPV-infected tissues of Spodoptera frugiperda larvae. We show that, in wild type virus, both proteins colocalize in fibrillar structures and associated electron-dense spacers which progress to encircle the polyhedra, as well as in completed polyhedron envelopes. In cells infected with polyhedrin-negative (PH-) viruses, an unusual proliferation of these spacers was observed suggesting a deregulatory event in the envelope assembly process. Results of Northern and Western blot analysis revealed that synthesis of P10 and calyx mRNA and proteins in PH- AcMNPV is unaffected as compared to wild type virus. Taken together, the observed physical and compositional connection between fibrillar structures, spacers and polyhedron envelopes, as well as the abnormal appearance of the spacers in PH- mutants, provide further evidence in support of a cooperative role of these structures in the assembly of the polyhedron envelope.

Animals↗
PubMed2004

Improving the insecticidal activity of Bacillus thuringiensis subsp. aizawai against Spodoptera exigua by chromosomal expression of a chitinase gene.

A transcriptionally fused gene comprising the P19 gene from Bacillus thuringiensis subsp. israelensis fused with a chitinase gene (chiBlA) from B. licheniformis was integrated into the B. thuringiensis subsp. aizawai BTA1 genome by homologous recombination. The resulting B. thuringiensis subsp. aizawai strain (INT1) showed growth and sporulation comparable with that of the wild-type strain. INT1 produced four chitinases of different molecular masses (i.e., 66, 55, 39, 36 kDa). Three of these (66, 55, 36 kDa) were derived from the cloned chiBlA gene, whereas the 39-kDa chitinase originated from BTA1. Using surface contamination bioassays, the 50% lethal concentration of lyophilized whole culture broth of INT1 against Spodoptera exigua neonate larvae was 12.2 microg/cm2, compared with 30.8 microg/cm2 for BTA1. Bioassays using filtered culture supernatant of INT1 (110 microg/cm2) together with trypsin-activated purified Cry1C protein of B. thuringiensis (1,280 ng/cm2) showed 75.0% mortality, compared with 56.7% mortality for Cry1C combined with BTA1 at the same concentration. Using scanning electron microscopy, clear perforations were observed in S. exigua fifth instar peritrophic membranes incubated with either crude or purified chitinase, or isolated from fifth instar S. exigua fed purified chitinase since the first instar. These results show that chitinase can increase the activity of B. thuringiensis subsp. aizawai against S. exigua. This is the first documentation of expressing a chimeric chitinase gene on the chromosome of B. thuringiensis; and chromosomal integration might be used as a potential technique for strain improvement.

Animals↗
PubMed2005

Antimicrobial activity of conditioned medium fractions from Spodoptera frugiperda Sf9 and Trichoplusia ni Hi5 insect cells.

Concentrated conditioned medium (CM) fractions from Spodoptera frugiperda Sf9 and Trichoplusia ni cells, eluting from a gel filtration column at around 10 kDa, were found to exhibit strong antibacterial activity against Bacillus megaterium and Escherichia coli. The B. megaterium cells incubated in the CM fraction from Sf9 cells rapidly lost viability: after 8 min the viability had decreased to 0.7%, as compared with the control. Addition of the CM fraction to E. coli cells resulted in a less drastic drop in viability: 65% viability was lost after 60 min of incubation. Further, exposure to the CM fraction caused a substantial leakage of intracellular proteins, as demonstrated by SDS-PAGE analysis. Cell lysis was confirmed by optical density measurements, microscopic investigations and flow cytometry. B. megaterium exposed to a CM fraction from T. ni cells lost 97% of their viability in about 40 min. Ubiquitin, thioredoxin and cyclophilin were identified in the antibacterial fraction from Sf9 cells by mass spectrometry and N-terminal amino acid sequencing. Other proteins in the fraction gave no matches in a database search. Since ubiquitin was shown not to cause the antimicrobial effect and thioredoxin and cyclophilin were likely not involved, the responsible agent may be an unknown protein, not yet registered in databases. The antimicrobial effect of the CM fraction from T. ni cells most probably comes from a lysozyme precursor protein.

Animals↗
PubMed2001

Establishment and characterization of cell-free translation/glycosylation in insect cell (Spodoptera frugiperda 21) extract prepared with high pressure treatment.

A coupled cell-free translation/glycosylation system, prepared from Spodoptera frugiperda insect cells, was established and optimized for protein production and glycosylation efficiency. Both translation and glycosylation were stimulated by addition of Mg2+, K+, ATP, GTP, creatine kinase and creatine phosphate, suggesting that glycoprotein productivity is largely determined by translation efficiency. However, high concentrations of creatine phosphate significantly inhibited translation. Spermidine stimulated both translation and glycosylation, but glycosylation required higher concentrations of spermidine than translation. Furthermore, extracts prepared at a nitrogen pressure of 10 kg/cm2 with the Mini-Bomb cell disruption chamber had the highest glycoprotein productivity; and extracts prepared at the higher nitrogen pressure of 15 kg/cm2 retained glycosylation ability. While extracts prepared with the Potter-Elvehjem homogenizer could mediate translation, no glycosylation was achieved. This indicated that the posttranslational machinery might survive disruption by high pressure, but not by physical shearing force. This insect cell-free system was able to synthesize approximately 25 microg of glycosylated gp120/ml of reaction mixture.

Animals↗
PubMed2001

Isolation and characterization of a Bacillus thuringiensis ssp. kurstaki strain toxic to Spodoptera exigua and Culex pipiens.

A strain of Bacillus thuringiensis with dual toxicity was isolated from Korean soil samples and named K2. K2 was determined as ssp. kurstaki (H3a3b3c) by serological test and produced bipyramidal-shaped parasporal inclusions. The plasmid and protein profiles of B. thuringiensis K2 were different from those of the reference strain, ssp. kurstaki HD-1. To verify gene type of B. thuringiensis K2, PCR analysis with specific cry gene primers was performed. The result showed that B. thuringiensis K2 had cry1Aa, cry1Ab, cry1C, and cry1D type genes, whereas ssp. kurstaki HD-1 had cry1Aa, cry1Ab, cry1Ac, and cry2 type genes. In addition, B. thuringiensis K2 had high toxicity against Spodoptera exigua and Culex pipiens, whereas B. thuringiensis ssp. kurstaki HD-1 does not have high toxicity against these two insect species.

Animals↗
PubMed1999

Mutations of loop 2 and loop 3 residues in domain II of Bacillus thuringiensis Cry1C delta-endotoxin affect insecticidal specificity and initial binding to Spodoptera littoralis and Aedes aegypti midgut membranes.

Site-directed mutagenesis was used to examine the role of predicted loops 2 (374QPWP377) and 3 (436QRSGTPF442) in domain II of the Bacillus thuringiensis Cry1C delta endotoxin for insecticidal specificity and receptor binding. Q374E, S438F, and G439A substitutions resulted in near or complete loss of toxicity toward both Spodoptera littoralis and Aedes aegypti. R437K, R437I, and G439V mutants exhibited significantly reduced toxicity to S. littoralis and A. aegypti, while mutations of T440, P441, and F442 showed only slight reductions in toxicity to both insects. Loop 2 mutations Q374N, P375A, W376Y, and P377A did not significantly affect S. littoralis toxicity but exhibited reduced activity to A. aegypti. In contrast, the loop 3 mutations Q436K, Q436E, and S438Y had no effect on A. aegypti toxicity, but showed significantly decreased S. littoralis activity. Heterologous competition binding assays with brush border membrane vesicles (BBMV) from both insects correlated well with the toxicity data with the exception of the R437 mutants, where steps other than initial receptor binding appear to be involved. Overall we conclude that, while loops 2 and 3 play an important role in binding and toxicity to both insects, loop 2 appears to play the greater role in A. aegypti activity, while loop 3 is more important for S. littoralis toxicity.

Aedes↗
PubMed2005

Interactions of mechanical stimuli and sex pheromone information in antennal lobe neurons of a male moth, Spodoptera littoralis.

Male moths respond to sex pheromone sources with up-wind flight behaviour. Localization of the odour source requires not only detection of the olfactory stimulus, but also other sensory input regarding, e.g. visual and mechanical stimuli. Thus, integration of different types of sensory input is necessary. It is, however, not known where in the central nervous system the integration of information regarding different sensory modalities takes place. Using intracellular recording and staining techniques, we investigated neurons in the antennal lobe of Spodoptera littoralis, during stimulation with a mechanical stimulus and a sex pheromone. Fifteen percent of all the neurons investigated responded to the mechanical stimulus and the majority of these neurons showed altered responses if the olfactory stimulus was added. A receptor neuron responding only to the wind stimulus was found to arborise in the antennal lobe. Most projection neurons responded with an enhanced action potential frequency to the combined stimulus. In local interneurons, enhancement, depression, or no change of the responses to the wind stimulus was found when the olfactory stimulus was added. The results suggest that neurons present in the antennal lobe integrate mechanosensory and olfactory input, possibly assisting the moths to orient during up-wind flight towards an odour source.

Action Potentials↗
PubMed2002

Quantitative relationships between induced jasmonic acid levels and volatile emission in Zea mays during Spodoptera exigua herbivory.

Jasmonic acid (JA) has long been hypothesized to be an important regulator of insect-induced volatile emission; however, current models are based primarily on circumstantial evidence derived from pharmacological studies. Using beet armyworm caterpillars (BAW: Spodoptera exigua) and intact corn seedlings, we examine this hypothesis by measuring both the time-course of insect-induced JA levels and the relationships between endogenous JA levels, ethylene, indole and sesquiterpenes. In separate Morning and Evening time-course trials, BAW feeding stimulated increases in JA levels within the first 4-6 h and resulted in maximal increases in JA, indole, sesquiterpenes and ethylene 8-16 h later. During BAW herbivory, increases in JA either paralleled or preceded the increases in indole, sesquiterpenes and ethylene in the Morning and Evening trials, respectively. By varying the intensity of the BAW herbivory, we demonstrate that strong positive relationships exist between the resulting variation in insect-induced JA levels and volatile emissions such as indole and the sesquiterpenes. To address potential signaling interactions between herbivore-induced JA and ethylene, plants were pretreated with 1-methylcyclopropene (1-MCP), an inhibitor of ethylene perception. 1-MCP pretreatment resulted in reduced production of ethylene and volatile emission following BAW herbivory but did not alter the insect-induced accumulation of JA. Our results strongly support a role for JA in the regulation of insect-induced volatile emission but also suggest that ethylene perception regulates the magnitude of volatile emission during herbivory.

Animals↗
PubMed2000

Accumulation of a maize proteinase inhibitor in response to wounding and insect feeding, and characterization of its activity toward digestive proteinases of Spodoptera littoralis larvae.

The mpi gene encodes a maize proteinase inhibitor (MPI) protein whose mRNA accumulates in response to mechanical wounding. In this study, mpi gene expression in response to different types of damage was investigated. In mechanically damaged leaves of maize (Zea mays L.), mpi mRNA accumulation was affected by the degree of damage inflicted on the leaf. Consecutive wounds resulted in higher levels of mpi transcripts. The MPI protein was expressed in Escherichia coli and purified. Polyclonal antibodies were then produced and used to study MPI accumulation in insect-wounded and mechanically wounded maize leaves. When larvae of the lepidopteran insect Spodoptera littoralis were fed on maize leaves, MPI accumulated in tissues adjacent to the wound site. The level of inhibitor accumulation was higher in leaves chewed by larvae than in leaves that had been damaged mechanically. Longer feeding periods also resulted in higher levels of MPI accumulation. Additionally, the inhibitory properties of MPI toward mammalian and insect digestive serine proteinases were determined. Contrary to the majority of the plant proteinase inhibitors described, MPI is an inhibitor of mammalian elastase that only weakly inhibits mammalian chymotrypsin. However, both elastase and chymotrypsin-like activities from the larval midgut of S. littoralis were effectively inhibited by MPI. We discuss these results with regard to the function and evolution of plant proteinase inhibitors. The availability of a plant proteinase inhibitor which is able to inhibit the two types of insect digestive proteinase, elastase and chymotrypsin, might be useful for engineering protection against lepidopteran insect pests in transgenic plants.

Amino Acid Sequence↗
PubMed1997

Enhancer element, repetitive sequences and gene organization in an 8-kbp region containing the polyhedrin gene of the Spodoptera littoralis nucleopolyhedrovirus.

The Spodoptera littoralis multicapsid nucleopolyhedrovirus (SpliMNPV) shows only a distant genetic relationship to other NPVs. In this report we describe the gene organization of an 8-kbp region of the SpliMNPV which contains the polyhedrin gene. The polyhedrin transcription initiation sites were mapped and the sequence and gene organization of an 8-kbp region of SpliMNPV were determined. The sequences downstream of the polyhedrin gene showed colinearity with the gene organization of other NPVs. An anticlockwise 1035-bp open reading frame (ORF), capable of encoding a proline-rich polypeptide, was found at the 3' end of the polyhedrin gene. followed by an 837-bp ORF encoding a putative protein kinase (PK), with an orientation similar to that of the polyhedrin gene. Sequences upstream of the polyhedrin gene were found to be unique to SpliMNPV and contained two regions consisting of highly repetitive sequences. One region, 980 bp in length and termed sequence repeat region 1 (SR1), contained a variety of short direct repeats, SR1 was found to act as a transcriptional enhancer in a transient expression assay. Additional regions containing different repetitive sequences were identified within the proline-rich ORF1035 and in sequences located downstream of the pk gene.

Amino Acid Sequence↗
PubMed1998

Identification and characterization of the Spodoptera littoralis nucleopolyhedrovirus type B lef-3 gene.

We have identified a gene from the Spodoptera littoralis nucleopolyhedrovirus type B (SpliNPV-B) with several characteristics that suggest that it is homologous to the lef-3 genes of the Autographa californica and Orgyia pseudotsugata NPVs (AcMNPV and OpMNPV, respectively). The SpliNPV-B lef-3 gene was mapped between 43.6 and 45.5 map units of the SpliNPV-B genome. Northern blot analysis showed that SpliNPV-B lef-3 was expressed as a 1.6 Kb transcript at 5 h post infection (p.i.), reached high levels at 24 h p.i., and remained highly expressed at 56 h p.i. Transcription of SpliNPV-B lef-3 initiated at two distinct sites downstream from a TATA-box motif and terminated 25 nucleotides downstream from the translation stop site of the putative LEF-3 polypeptide. The 5'-boundaries of lef-3 promoter elements were investigated by transient expression assays, which revealed that the major components of the lef-3 promoter are within a 183 base pair region upstream of the distal transcription initiation site. Transfection of SpliNPV-B infected Sf9 cells with anti-sense oligonucleotides designed to inhibit LEF-3 expression resulted in substantial reduction of viral DNA replication, suggesting that the role of SpliNPV-B lef-3 may be similar to that of AcMNPV and OpMNPV lef-3 genes, which are essential for viral DNA replication.

Amino Acid Sequence↗
PubMed2000

Characteristics of the transactivator gene ie1 of Spodoptera exigua multiple nucleopolyhedrovirus.

The genomic position and nucleotide sequence of the immediate early gene ie1 of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) were determined. The SeMNPV ie1 gene had the potential to encode a protein of 714 amino acids with a predicted molecular mass of 82.0 kDa, representing the largest baculovirus IE1 known to date. The similarity of SeMNPV IE1 with IE1 proteins from other baculoviruses was restricted to the basic C-terminal two-thirds of the protein, which is involved in DNA binding. A single ie1 transcript of 2.5 kb was detected at 1 h p.i., peaking at 8 h p.i. and disappearing at 24 h p.i. From 8 h p.i. onwards a 1.7 kb transcript was detected, which became more abundant late in infection. Primer extension analysis revealed the use of several start sites early and late in infection. One of these was located in the promoter motif CAGT located at position -15 relative to the translational start codon, most intensively used at 8 h p.i. Four kb upstream of the SeMNPV ie1 gene, ORF xd244 was identified with homology to the used left ie0 exon of AcMNPV, OpMNPV and LdMNPV. Phylogenetic analysis indicated that the SeMNPV IE1 protein shared the most recent ancestor with its HzSNPV and LdMNPV homologues, although their IE1 proteins had diverged considerably from each other and from other baculoviruses.

Amino Acid Sequence↗
PubMed2001

Identification, transcription and sequence analysis of the Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) DNA polymerase gene.

Sequence analysis of a 6.4 kb DNA region from the Spodoptera littoralis multinucleocapsid nucleopolyhedrovirus (SpliNPV) revealed a large open reading frame (ORF) encoding a predicted polypeptide of 998 amino acid (aa) residues with a molecular mass of 114.93 kDa, located between 47.2-52.3 m.u. on the SpliNPV genome. Comparative sequence analyses demonstrated that the ORF encodes a DNA polymerase gene (dnapol) that contains conserved exonuclease domains and DNA polymerase motifs found in many prokaryotic, eukaryotic, and viral replicative DNA polymerases. A second ORF, ORF138, located between the lef-3 and dnapol, encodes a 138 aa polypetide that is homologous to ORF66 of the Autographa californica MNPV (AcMNPV). SpliNPV DNA polymerase shares an overall aa sequence identity of 39% with that of AcMNPV. A 3.0 kb SpliNPV dnapol-specific transcript was detected initially at 2 hpi and became abundant 48 hpi by Northern blot analysis. The transcription initiation site was mapped to an NPV early promoter element, ACGT. 3' RACE demonstrated that the SpliNPV dnapol transcript terminated at the polyadenylation signal AATAAA. Sequence analysis suggested that the SpliNPV dnapol and the dnapol of the NPV of S. litura (SpltNPV) are closely related.

Amino Acid Sequence↗
PubMed2006

Stable RNA interference in Spodoptera frugiperda cells by a DNA vector-based method.

Double-stranded RNA (dsRNA)-mediated interference (RNAi) is a powerful tool for silencing of gene expression in many organisms. To establish a DNA vector-based method for stable RNAi in Spodoptera frugiperda cells (Sf9), we created a stably transfected Sf9 cell line to express large dsRNA fragment targeting to silence the firefly luciferase gene (luc). The luc dsRNA specifically and stably suppressed the baculovirus-mediated luciferase expression. Thus, gene silencing in Sf9 cells was achieved using DNA vectors similar to the facile design described in this study.

Animals↗
PubMed2006

Isolation, identification, synthesis, and field evaluation of the sex pheromone of the Brazilian population of Spodoptera frugiperda.

Several studies have shown intraspecific geographical variation in the composition of sex pheromones. Pheromone lures from North America and Europe were not effective against the fall armyworm Spodoptera frugiperda (Smith, 1797) (Lepidoptera: Noctuidae) in Brazil, so we examined the composition of the sex pheromone produced by females from Brazilian populations. Virgin female gland extracts contained (Z)-7-dodecenyl acetate (Z7-12:Ac), (E)-7-dodecenyl acetate (E7-12:Ac), dodecyl acetate, (Z)-9-dodecenyl acetate, (Z)-9-tetradecenyl acetate (Z9-14:Ac), (Z)-10-tetradecenyl acetate, tetradecyl acetate/(Z)-11-tetradecenyl acetate (Z11-16:Ac), and (Z)-11-hexadecenyl acetate. The relative proportions of each acetate were 0.8:1.2:0.6:traces:82.8:0.3:1.5:12.9, respectively. This is the first time that E7-12:Ac has been reported from the pheromone gland of S. frugiperda. Only three compounds, Z9-14:Ac, Z7-12:Ac, and E7-12:Ac, elicited antennal responses, and there were no differences in catch between traps baited with either Z7-12:Ac + Z9-14:Ac or Z7-12:Ac + Z9-14:Ac + Z11-16:Ac blends. However, the Z7-12:Ac + Z9-14:Ac + E7-12:Ac blend was significantly better than Z7-12:Ac + Z9-14:Ac, indicating that E7-12:Ac is an active component in the sex pheromone of the Brazilian populations of S. frugiperda.

Animals↗
PubMed2006

Attraction of Spodoptera frugiperda larvae to volatiles from herbivore-damaged maize seedlings.

Plants respond to insect attack with the induction of volatiles that function as indirect plant defenses through the attraction of natural enemies to the herbivores. Despite the fact that volatiles are induced in response to caterpillar attack, their reciprocal effects on the host location behaviors of the same foraging herbivores are poorly understood. We examined orientation responses of sixth instar fall armyworm [FAW; Spodoptera frugiperda (Smith)] to odors from herbivore-damaged and undamaged maize seedlings (Zea mays var. Golden Queen) in y-tube olfactometer bioassays. While both damaged and undamaged maize seedlings were attractive compared with air, sixth instars preferred odors from damaged maize seedlings over odors from undamaged maize seedlings. Gas chromatography-mass spectrometry analysis of plant volatiles revealed that linalool and 4,8-dimethyl-1,3,7-nonatriene were the major volatiles induced by FAW herbivory 6 hr after initial damage. Given its prominence in induced plants and established attractiveness to adult FAW, linalool was evaluated both as an individual attractant and as a supplemental component of whole plant odors. Volatile linalool was more attractive than air to sixth instar FAW over a broad range of release rates. FAW also responded selectively to different amounts of linalool, preferring the higher amount. The orientation preferences of FAW were readily manipulated through capillary release of linalool into the airstream of whole plant odors. FAW preferred linalool over undamaged plant odors, and linalool-supplemented plant odors over unsupplemented plant odors, indicating that olfactory preferences could be changed by alteration of a single volatile component. These results suggest that although many induced volatiles attract natural enemies of herbivores, these defenses may also inadvertently recruit more larval herbivores to an attacked plant or neighboring conspecifics.

Animals↗
PubMed2006

Altered glucosinolate hydrolysis in genetically engineered Arabidopsis thaliana and its influence on the larval development of Spodoptera littoralis.

The antiherbivore potential of the glucosinolate-myrosinase defense system found in plants of the order Capparales is heavily influenced by the types of hydrolysis products (e.g. isothiocyanates, nitriles) formed from the parent glucosinolates upon plant damage. However, comparison of the effects of glucosinolate hydrolysis products on insect herbivores has been hampered by the lack of suitable experimental tools for rigorous bioassays, such as intact plants differing only in the types of hydrolysis products they produce, or artificial diets that can accurately simulate glucosinolate hydrolysis. The wide array of molecular resources for Arabidopsis thaliana has facilitated the identification of several genes that play a role in glucosinolate hydrolysis. One of these encodes the epithio-specifier protein (ESP) that promotes the formation of nitriles at the expense of isothiocyanates in certain ecotypes of A. thaliana. We overexpressed the ESP cDNA from the nitrile-producing ecotype Landsberg erecta in the isothiocyanate-producing ecotype Columbia-0 to generate transgenic lines of A. thaliana that differed from wild-type plants in the type of glucosinolate hydrolysis products formed upon tissue damage, whereas parent glucosinolate profile and myrosinase activity levels, as well as plant morphology and growth habit, remained unchanged. Bioassays with the model generalist herbivore Spodoptera littoralis (Lepidoptera: Noctuidae) demonstrated that larvae reared on the nitrile-producing lines on average gained weight faster in the first larval stages than larvae that fed on isothiocyanate-producing control plants. Furthermore, larvae with medial growth rates showed a tendency to pupate earlier on the ESP-overexpressing plant lines. Together with the results of previous studies, these findings suggest that isothiocyanates are more effective defenses against insect herbivores than nitriles, and raise questions about what conditions select for nitrile formation in plants.

Amino Acid Sequence↗
PubMed2004

Identification and expression of a conserved ubiquitin gene homologue of Spodoptera litura nucleopolyhedrovirus (spltNPV-I).

An ORF having a potential to code for a polypeptide of 79 amino acids has been identified within 993 nt sequence of 2 kb EcoRI-W fragment of Spodoptera litura nucleopolyhedrovirus (SpltNPV-I). Nucleotide and deduced amino acid sequence analyses showed its identity with the ubiquitin homologue of eukaryotes (79-80%), Melanoplus sanguinipes entomopoxvirus (76%) and other baculoviruses (72-89%). The ORF is under baculovirus late promoter motif RTAAG but unlike other baculoviruses, three such motifs at -6, -10 and -27 position are present in SpltNPV. The ORF expresses as a 10 kDa proteinin E. coli and the purified recombinant protein showed crossreactivity with the rabbit anti-ubiquitin antibodies.

Amino Acid Sequence↗