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PubMed1990

Characteristically distinct isolates of the nuclear polyhedrosis virus from Spodoptera litura.

More than 100 isolates were plaque-purified to examine the genetic variations in four wild stocks of Spodoptera litura nuclear polyhedrosis virus (NPV) collected in Japan. These isolates were characterized by their in vitro host range in three established insect cell lines, growth characteristics, polyhedral protein, DNA restriction endonuclease pattern and DNA hybridization. The isolates were separated into four distinct groups: (I) isolates corresponding to Autographa californica NPV, (II and IV) two different groups of isolates of S. littoralis NPV which had been previously characterized and (III) isolates with no correspondence to any reported virus group. Of the S. litura NPV wild stocks, two were mixtures of more than two different groups of NPVs. We have discussed the advantage of having a mixture of different NPV groups in the same wild virus stocks.

Animals↗

PubMed1990

Expression of avian leukaemia virus env-gp85 in Spodoptera frugiperda cells by use of a baculovirus expression vector.

We studied the genetic expression of gp85 of avian leukaemia virus (ALV) subgroup A in a baculovirus/insect cell system. 5'terminal sequences of the gag gene were added to precede the ALV gp85 sequence and a stop codon was introduced at the boundary of gp85 and gp37. The resulting construct was then cloned into the baculovirus transfer vector pAcYM1, which contains the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). Cells of the insect Spodoptera frugiperda (Sf9) were cotransfected with the resulting recombinant transfer vector pAc85 and infectious AcNPV/E2 DNA. After cotransfection, recombinant baculovirus that lacked the polyhedrin gene and expressed gp85 was selected from the supernatant and used to infect Sf9 cells. The expression of the gp85 gene peaked 3 days after infection, but expression products were not released into the culture medium even though the signal peptide had been cleaved. Owing to incomplete N-glycosylation in the insect cells the largest gp85 product had an Mr of only 65,000. In immunofluorescence tests and immunoblots the recombinant gp85 products reacted with polyclonal and monoclonal antibodies directed against ALV gp85 of subgroup A. Chickens inoculated with crude lysates of Sf9 cells infected with gp85-expressing recombinant baculovirus developed antibodies directed against ALV gp85. These antibodies were not capable of neutralizing ALV.

Animals↗

PubMed1991

Baculovirus-directed high level expression of the hepatitis delta antigen in Spodoptera frugiperda cells.

The hepatitis delta antigen (HDAg) is a multifunctional protein. It forms the core-like structure of the hepatitis delta virus (HDV) but also enhances replication of HDV in the nucleus of the hepatocyte. A cDNA fragment encoding HDAg was inserted adjacent to the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus present in the baculovirus transfer vector pVL941. After transfection of Spodoptera frugiperda (Sf9) cells a recombinant baculovirus Ac delta 1 was isolated and purified using filter hybridization techniques. Sf9 cells infected with Ac delta 1 express the HDAg as a non-fused, non-glycosylated protein with an abundance of up to 25% of the total cellular protein mass. Immunoblot analysis using a human polyclonal anti-HD conjugate identified a 22K and a 24K protein in the nucleus of Ac delta 1-infected Sf9 cells. Electron microscopic studies using immunogold labelling showed that the recombinant HDAg (recHDAg) was associated with the hetero-chromatin of the Sf9 cells. The recHDAg produced by Sf9 cells elicited anti-HD antibodies in chimpanzees when injected intramuscularly.

Animals↗

PubMed1991

Characterization of yellow fever virus proteins E and NS1 expressed in Vero and Spodoptera frugiperda cells.

The cDNA encoding the E and NS1 proteins of the yellow fever virus (YFV) was expressed in Spodoptera frugiperda cells via the recombinant baculovirus Ac-E. NS1 as a gp100 precursor which was cleaved to generate the recombinant proteins E and NS1 similar in size, folding and antigenicity to the authentic ones. Recombinant protein E exhibited immunodominant epitopes as judged by its reactivity with YFV-neutralizing MAbs. Using the Triton X-114 phase separation system, authentic and recombinant E proteins as well as the gp100 precursor exhibited hydrophobic properties similar to those of integral membrane proteins. Recombinant protein E was found neither in the extracellular medium nor on the cell surface, suggesting that it did not migrate within the secretory pathway of insect cells. Analysis of protein NS1 expressed in primate and insect cells revealed that the newly synthesized 48K NS1 glycoprotein was converted to a heat-labile gp72 homo-oligomeric form. This phenomenon did not require the presence of carbohydrate groups. Using the Triton X-114 phase separation system, the oligomeric form of NS1 was shown to be associated with cellular membranes although it appeared less hydrophobic than protein E and gp100. A small fraction of YFV NS1 oligomers were transported throughout the secretory pathway to be shed into the extracellular medium of primate cells. YFV NS1 oligomers migrated from the endoplasmic reticulum to the Golgi complex, whereas their N-oligosaccharides of the high-mannose type are processed to the complex-mannose type. Protein NS1 expressed by recombinant baculovirus-infected insect cells was not found in the extracellular medium but associated with the plasma membrane of the cells. Two recombinant NS1 forms were detected in insect cells: a major one with an apparent Mr of 48K and a minor one of 47K in which N-linked glycans were probably processed to a trimannosyl core without further elongation. Thus, it appears that the transport strategy as well as the N-glycosylation of NS1 in insect cells infected with recombinant baculovirus were different from those of the NS1 in primate cells infected with YFV.

Animals↗

PubMed1992

Nucleotide sequence and transcriptional analysis of the polyhedrin gene of Spodoptera exigua nuclear polyhedrosis virus.

The nucleotide sequence of a 1.1 kbp fragment of the multiple nucleocapsid nuclear polyhedrosis virus (MNPV) of Spodoptera exigua (Se) containing the polyhedrin gene was determined. An open reading frame (ORF) of 738 nucleotides (nt) was detected. This ORF encoded a protein of 246 amino acids with a predicted M(r) of 29K. The nucleotide and amino acid sequences were compared with the sequences of eight other NPV polyhedrins. The SeMNPV polyhedrin protein was most closely related to S. frugiperda MNPV polyhedrin with differences in only five amino acids, and most distantly related to the Lymantria dispar MNPV polyhedrin. The size of the mRNA was approximately 1,000 nt, as determined by Northern blot analysis. Using primer extension assays and S1 nuclease mapping the transcriptional start and stop sites of the polyhedrin mRNA were located. The 5' regulatory sequence appeared to be 44 nt in length with the mRNA start site predominantly at the first A of the TAAG consensus start sequence. Two degenerate poly(A) signals were found immediately downstream of the translational stop signal. The transcriptional stop was located approximately 230 nt downstream from the translational stop signal, in an AT-rich sequence that appears to be common to all baculovirus polyhedrin genes. The SeMNPV polyhedrin mRNA does not appear to be polyadenylated.

Amino Acid Sequence↗

PubMed1993

Nucleotide sequence and transcriptional analysis of the p10 gene of Spodoptera exigua nuclear polyhedrosis virus.

The p10 gene of Spodoptera exigua multiple nuclear polyhedrosis virus (SeMNPV) was localized on the XbaI fragment H (5.1 kb) of the physical map of the viral genome. The coding sequence of the SeMNPV p10 gene is 264 nucleotides (nt) long corresponding to a predicted protein of 88 amino acids with an MHF of 9607. The SeMNPV p10 protein showed only limited amino acid identity (39% and 26%, respectively) to those of Orgyia pseudotsugata MNPV (OpMNPV) and Autographa californica MNPV (AcMNPV) and thus appears less conserved than other viral proteins. The SeMNPV p10 gene was expressed by a transcript of approximately 450 nt, which started in the conserved baculovirus late gene promoter motif TAAG. The leader of the SeMNPV p10 transcript was AT-rich (92%) and at 36 nt was the shortest leader of all baculovirus major late genes reported so far. The SeMPNV p10 transcript terminated 6 nt downstream from a putative poly(A) signal sequence (AATAAA); the latter was 61 nt downstream of the translational stop codon TAA. Upstream and downstream of the p10 gene, partial putative ORFs were found that showed significant amino acid sequence identity to the baculovirus p26 and p74 proteins. It is concluded that the region of SeMNPV DNA containing the p10 gene is collinear with the corresponding regions in the AcMNPV and OpMNPV genomes.

Amino Acid Sequence↗

PubMed1998

Opposite effects on spodoptera littoralis larvae of high expression level of a trypsin proteinase inhibitor in transgenic plants

This work illustrates potential adverse effects linked with the expression of proteinase inhibitor (PI) in plants used as a strategy to enhance pest resistance. Tobacco (Nicotiana tabacum L. cv Xanthi) and Arabidopsis [Heynh.] ecotype Wassilewskija) transgenic plants expressing the mustard trypsin PI 2 (MTI-2) at different levels were obtained. First-instar larvae of the Egyptian cotton worm (Spodoptera littoralis Boisd.) were fed on detached leaves of these plants. The high level of MTI-2 expression in leaves had deleterious effects on larvae, causing mortality and decreasing mean larval weight, and was correlated with a decrease in the leaf surface eaten. However, larvae fed leaves from plants expressing MTI-2 at the low expression level did not show increased mortality, but a net gain in weight and a faster development compared with control larvae. The low MTI-2 expression level also resulted in increased leaf damage. These observations are correlated with the differential expression of digestive proteinases in the larval gut; overexpression of existing proteinases on low-MTI-2-expression level plants and induction of new proteinases on high-MTI-2-expression level plants. These results emphasize the critical need for the development of a PI-based defense strategy for plants obtaining the appropriate PI-expression level relative to the pest's sensitivity threshold to that PI.

Journal Article↗

PubMed2004

Salicylic acid inhibits jasmonic acid-induced resistance of Arabidopsis thaliana to Spodoptera exigua.

The role of salicylic acid (SA) in plant responses to pathogens has been well documented, but its direct and indirect effects on plant responses to insects are not so well understood. We examined the effects of SA, alone and in combination with jasmonic acid (JA), on the performance of the generalist herbivore, Spodoptera exigua, in wild-type and mutant Arabidopsis thaliana genotypes that varied genetically in their ability to mount SA- and JA-mediated defence responses. In one experiment, growth of S. exigua larvae was highest on the Wassilewskija wild-type, intermediate on the Columbia wild-type and the JA-deficient fad mutant, and lowest on the nim1-1 and jar1-mutants, which are defective in the SA and JA pathways, respectively. Activity of guaiacol peroxidase, polyphenoloxidase, n-acetylglucosaminidase, and trypsin inhibitor varied by genotype but did not correlate with insect performance. SA treatment increased growth of S. exigua larvae by approximately 35% over all genotypes, but had no discernable effect on activities of the four defence proteins. In a second experiment, growth of S. exigua was highest across treatments on the cep1 mutant, a constitutive expressor of high SA levels and systemic acquired resistance, and lowest on the fad mutant, which is JA-deficient. JA treatment generally increased activity of all four defence proteins, increased total glucosinolate levels and reduced insect growth by approximately 25% over all genotypes. SA generally inhibited expression of JA-induced resistance to S. exigua when both hormones were applied simultaneously. Across genotypes and treatments, larval mass was negatively correlated with the activity of trypsin inhibitor and polyphenoloxidase and with total glucosinolate levels, and insect damage was negatively correlated with the activity of polyphenoloxidase. SA had little effect on the induction of defence protein activity by JA. However, SA attenuated the induction of glucosinolates by JA and therefore may explain better the interactive effects of SA and JA on insect performance. This study illustrates that direct and indirect cross-effects of SA on resistance to S. exigua can occur in A. thaliana. Effects of SA may be mediated through effects on plant defence chemistry or other aspects of the suitability of foliage for insect feeding and growth.

Acetylglucosaminidase↗

PubMed1989

Nucleotide sequence and analysis of the N-terminal coding region of the Spodoptera-active delta-endotoxin gene of Bacillus thuringiensis aizawai 7.29.

The nucleotide sequence of a 2711bp DNA segment which contains the N-terminal coding sequence and the 5' flanking region of a crystal protein gene (bta) from Bacillus thuringiensis subsp. aizawai 7.29 has been determined. The coding region encodes an 824 amino-acid polypeptide corresponding to a carboxy-terminally truncated delta-endotoxin specifically active against the cotton leaf worm Spodoptera littoralis. Comparison of the deduced amino acid sequence of the bta gene with that of the 4.5, 5.3 and 6.6 kb classes of lepidopteran-active delta-endotoxins revealed that the Bta sequence contains a very high level of amino acid substitutions in the N-terminal part of the protoxin molecule. The substitutions are grouped in several highly variable segments separated by highly conserved regions. These conserved domains are also present in the dipteran- and coleopteran-active delta-endotoxins. The control region of the bta gene shows considerable DNA identity with the control regions of the other lepidopteran-active genes. Deletions of the 3' region of the gene were carried out and the toxic fraction of the bta delta-endotoxin was identified with the N-terminal half of the molecule.

Amino Acid Sequence↗

PubMedDate not supplied

Complete sequence and gene organization of the Nosema spodopterae rRNA gene.

By sequencing the entire ribosomal RNA (rRNA) gene of Nosema spodopterae, we show here that its gene organization follows a pattern similar to the Nosema type species, Nosema bombycis, i.e. 5'-large subunit rRNA (2,497 bp)-internal transcribed spacer (185 bp)-small subunit rRNA (1,232 bp)-intergenic spacer (277 bp)-5S rRNA (114 bp)-3'. Gene sequences and the secondary structures of large subunit rRNA, small subunit rRNA, and 5S rRNA are compared with the known corresponding sequences and structures of closely related microsporidia. The results suggest that the Nosema genus may be heterogeneous and that the rRNA gene organization may be a useful characteristic for determining which species are closely related to the type species.

Animals↗

PubMed1988

Identification of a delta-Endotoxin Gene Product Specifically Active against Spodoptera littoralis Bdv. among Proteolysed Fractions of the Insecticidal Crystals of Bacillus thuringiensis subsp. aizawai 7.29.

At least three different insecticidal crystal protein genes were shown to be expressed in Bacillus thuringiensis subsp. aizawai 7.29, a strain that is potentially active against the cotton leafworm Spodoptera littoralis Bdv. Among crude K-60 fractions (60- to 70-kilodalton [kDa] molecules) that were products of proteolysed crystals containing the active domains of the protoxin molecules, we were able to distinguish several distinct components on the basis of their antigenic relationship and their larvicidal properties. A purified fraction designated SF2 was a 61-kDa component specifically active against Pieris brassicae L. and homologous to the B. thuringiensis subsp. berliner 1715 plasmid-encoded crystal protein. A second fraction designated SF1 was composed of 63- and 65-kDa polypeptides and was specifically active against S. littoralis. The SF1 fraction and particularly the 65-kDa component were not antigenically related to the 61-kDa component. The purified fractions were compared with the products of three different crystal protein genes we previously cloned from total DNA of B. thuringiensis subsp. aizawai, among them a new type of crystal protein gene encoding a protein that is specifically active against S. littoralis and other insects of the Noctuidae family. This approach led us to consider the 65-kDa component as a minimum active part of a delta-endotoxin that is encoded by this new gene. Products of the two other cloned genes can be correlated with the 61- and 63-kDa components, respectively. Thus, in B. thuringiensis subsp. aizawai 7.29, multiple delta-endotoxin genes of different structural types direct the synthesis of several delta-endotoxins with different host specificities which were identified as components of the insecticidal crystals.

Journal Article↗

PubMed1990

Plasmid transfer between strains of Bacillus thuringiensis infecting Galleria mellonella and Spodoptera littoralis.

To determine the possibility of plasmid transfer occurring between strains of Bacillus thuringiensis in infected lepidopterous larvae, Galleria mellonella and Spodoptera littoralis were infected with two or more strains of B. thuringiensis and the resulting bacteria from the dead insects were examined for plasmid transfer. Transfer rates of plasmids coding for crystal production and tetracycline resistance were high, reaching levels similar to those obtained in laboratory broth cultures. Transfer was higher in G. mellonella than S. littoralis, probably due to the greater ability of B. thuringiensis to colonize the larvae. In broth cultures, B. thuringiensis was also able to transfer plasmids into sporeforming bacteria present in soil samples. The results suggest that plasmid transfer between strains of B. thuringiensis occurs in nature, resulting in the production of new combinations of delta-endotoxins within populations of the bacteria.

Animals↗

PubMed1990

Toxicity to Spodoptera exigua and Trichoplusia ni of individual P1 protoxins and sporulated cultures of Bacillus thuringiensis subsp. kurstaki HD-1 and NRD-12.

The toxicities to neonate Spodoptera exigua and Trichoplusia ni of lyophilized powders obtained from sporulated liquid cultures (referred to as sporulated cultures) and Escherichia coli-expressed P1 [cryIA(a) cryIA(b) cryIA(c)] protoxins from three-gene strains of NRD-12 and HD-1 of Bacillus thuringiensis subsp. kurstaki were determined by using diet incorporation bioassays. Although sporulated cultures from both strains were more toxic to T. ni than S. exigua, there were no differences in toxicity between NRD-12 and HD-1. Toxicities of the three individual P1 protoxins against S. exigua varied by at least fivefold, with the cryIA(b) protein being the most toxic. These same protoxins varied in toxicity against T. ni by at least 16-fold, with the cryIA(c) protein being the most toxic. However, when tested against either S. exigua or T. ni, there were no differences in toxicity between an NRD-12 P1 protoxin and the corresponding HD-1 P1 protoxin. Comparing the toxicities of individual protoxins with that of sporulated cultures demonstrates that no individual protoxin was as toxic to S. exigua as the sporulated cultures. However, this same comparison against T. ni shows that both the cryIA(b) and cryIA(c) proteins are at least as toxic as the sporulated cultures. Results from this study suggest that NRD-12 is not more toxic to S. exigua than HD-1, that different protein types have variable host activity, and that other B. thuringiensis components are not required for T. ni toxicity but that other components such as spores might be required for S. exigua toxicity.

Animals↗

PubMed1995

Development of Bacillus thuringiensis CryIC Resistance by Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae).

Selection of resistance in Spodoptera exigua (Hubner) to an HD-1 spore-crystal mixture, CryIC (HD-133) inclusion bodies, and trypsinized toxin from Bacillus thuringiensis subsp. aizawai and B. thuringiensis subsp. entomocidus was attempted by using laboratory bioassays. No resistance to the HD-1 spore-crystal mixture could be achieved after 20 generations of selection. Significant levels of resistance (11-fold) to CryIC inclusion bodies expressed in Escherichia coli were observed after seven generations. Subsequent selection of the CryIC-resistant population with trypsinized CryIC toxin resulted, after 21 generations of CryIC selection, in a population of S. exigua that exhibited only 8% mortality at the highest toxin concentration tested (320 (mu)g/g), whereas the 50% lethal concentration was 4.30 (mu)g/g for the susceptible colony. Insects resistant to CryIC toxin from HD-133 also were resistant to trypsinized CryIA(b), CryIC from B. thuringiensis subsp. entomocidus, CryIE-CryIC fusion protein (G27), CryIH, and CryIIA. In vitro binding experiments with brush border membrane vesicles showed a twofold decrease in maximum CryIC binding, a fivefold difference in K(infd), and no difference in the concentration of binding sites for the CryIC-resistant insects compared with those for the susceptible insects. Resistance to CryIC was significantly reduced by the addition of HD-1 spores. Resistance to the CryIC toxin was still observed 12 generations after CryIC selection was removed. These results suggest that, in S. exigua, resistance to a single protein is more likely to occur than resistance to spore-crystal mixtures and that once resistance occurs, insects will be resistant to many other Cry proteins. These results have important implications for devising S. exigua resistance management strategies in the field.

Journal Article↗

PubMed1997

Interaction between Calcium Ions and Bacillus thuringiensis Toxin Activity against Sf9 Cells (Spodoptera frugiperda, Lepidoptera).

The effects of calcium ions and modulators of calcium movement on Bacillus thuringiensis insecticidal protein toxicity were investigated with Sf9 cells (Spodoptera frugiperda, fall armyworm) by a new B. thuringiensis toxicity assay based on measurement of fluorescence of ethidium homodimer, a high-affinity DNA stain. CryIC toxicity was substantially stimulated by extracellular calcium in a dose-dependent way (in the millimolar range), while toxicity enhancement could not be replicated when calcium was replaced by barium. This incremental toxicity was reduced by cobalt and lanthanum ions, two inorganic-calcium transport inhibitors. Methoxyverapamil, a voltage-dependent calcium channel blocker, and nifedipine, an inhibitor of dihydropyridine-sensitive L-type calcium channels, had no effect on CryIC toxin activity, but BAY K 8644, an L-type calcium channel activator, increased CryIC activity at high concentrations of extracellular calcium. While A23187, a calcium ionophore, and TMB-8, an inhibitor of intracellular-calcium mobilization, did not change CryIC-induced mortality, thapsigargin, an inhibitor of calcium uptake in intracellular stores, and more particularly trifluoperazine, which inhibits calcium-calmodulin-dependent processes, increased CryIC-mediated toxicity. The incremental effect of extracellular calcium on CryIC-induced toxicity was consistent with an increased concentration of intracellular calcium.

Journal Article↗

PubMed1998

Changes in Permeability of Brush Border Membrane Vesicles from Spodoptera littoralis Midgut Induced by Insecticidal Crystal Proteins from Bacillus thuringiensis.

Bacillus thuringiensis insecticidal crystal proteins (ICPs) are thought to induce pore formation in midgut cell membranes of susceptible insects. Cry1Ca, which is significantly active in Spodoptera littoralis, made brush border membrane vesicles permeable to KCl (osmotic swelling was monitored by the light scattering technique); the marginally active ICPs Cry1Aa, Cry1Ab, and Cry1Ac did not.

Journal Article↗

PubMed1975

Replication of nuclear polyhedrosis virus in a continuous cell culture of Spodoptera frugiperda: microscopy study of the sequence of events of the virus infection.

A microscopy study of the sequence of morphogenic events of Spodoptera frugiperda nuclear polyhedrosis virus infection of S. frugiperda cells is presented which orders the sequence of replication and establishes the time scale within which the events occur. The virus entered the cell by 1 h postinfection and was uncoated. The eclipse period was 9 h and the latent period was 12 h. Polyhedron formation was detected by 18 h postinfection and continued until the deposition of the polyhedron membrane was completed by 48 h postinfection. Aberrant morphogenic characteristics of virus repeatedly passaged in the cell culture were also recorded. Adsorption, envelope morphogenesis, and release mechanisms are discussed in light of other data on in vivo and in vitro baculovirus infections.

Adsorption↗

PubMed1981

alpha-Amanitin-Resistant Viral RNA Synthesis in Nuclei Isolated from Nuclear Polyhedrosis Virus-Infected Heliothis zea Larvae and Spodoptera frugiperda Cells.

[(3)H]RNA was synthesized in nuclei isolated at various times postinfection from the fat bodies of Heliothis zea larvae infected with H. zea nuclear polyhedrosis virus and from cultured Spodoptera frugiperda cells infected with Autographa californica nuclear polyhedrosis virus. To detect virus-specific RNA synthesis, the [(3)H]RNA was hybridized to denatured viral DNA immobilized on nitrocellulose filters. Nuclear polyhedrosis virus-specific RNA synthesis in the infected nuclei isolated from H. zea larval fat bodies and S. frugiperda cells was only inhibited 20 to 25% by concentrations of alpha-amanitin sufficient to inhibit the host RNA polymerase II. In addition, a productive nuclear polyhedrosis virus infection was obtained in S. frugiperda cells grown in the presence of an alpha-amanitin concentration that inhibited 90% of the cellular RNA polymerase II activity. The cellular RNA polymerase II enzyme remained sensitive to alpha-amanitin during infection, and there was no evidence that a virus-coded, alpha-amanitin-resistant enzyme was synthesized after the onset of infection. The data suggest that the bulk of nuclear polyhedrosis virus-specific RNA synthesis in isolated nuclei is transcribed by an enzyme other than the host RNA polymerase II.

Journal Article↗