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PubMed1981

Spodoptera frugiperda Nuclear Polyhedrosis Virus Genome: Physical Maps for Restriction Endonucleases BamHI and HindIII.

The physical map for the genome of Spodoptera frugiperda nuclear polyhedrosis virus was constructed for restriction endonucleases BamHI and HindIII. The ordering of the restriction fragments was accomplished by cross-blot hybridization of BamHI, HindIII, and EcoRI fragments. The alignment of the HindIII fragments within the BamHI map was achieved by double digestion with the two restriction endonucleases followed by cross-blot hybridization. The results showed that the viral genome consisted of mainly unique sequences. In addition, the circular nature of the viral genome was reaffirmed.

Journal Article↗
PubMed1982

Capping of Viral RNA in Cultured Spodoptera frugiperda Cells Infected with Autographa californica Nuclear Polyhedrosis Virus.

Viral RNA from fall armyworm (Spodoptera frugiperda) cells infected with Autographa californica nuclear polyhedrosis virus contains cap structures. Most of the cap labeled in vivo with [(3)H]methionine or (32)P(i) cochromatographed on DEAE-cellulose with the -5 charge marker; a minor component appeared at -4 net charge. The former is probably a cap 1 structure (m(7)GpppX(m) (p)Yp), and the latter is probably a cap 0 (m(7)GpppXp). On the basis of relative labeling of the two caps with [(3)H]adenosine and [(3)H]guanosine, we concluded that each cap contained at least one adenosine residue in addition to guanosine and, therefore, that cap 0 contained m(7)GpppAp. Cleavage of [(3)H]methionine-labeled viral RNA with tobacco acid pyrophosphatase released a labeled component that cochromatographed on polyethyleneimine-cellulose with m(7)Gp, confirming the m(7)GpppX linkage in the cap. These results were also seen with host polyadenylated RNA. The caps appeared in nearly equal abundance in viral polyadenylated and non-polyadenylated RNAs. The ratio of (32)P(i) incorporated into the cap to that incorporated into mononucleotides suggested average lengths for the polyadenylated and non-polyadenylated RNAs of 1,800 and 1,200 nucleotides, respectively.

Journal Article↗
PubMed1983

Viral Transcription During Autographa californica Nuclear Polyhedrosis Virus Infection: a Novel RNA Polymerase Induced in Infected Spodoptera frugiperda Cells.

Autographa californica nuclear polyhedrosis virus-specific RNA synthesis in isolated nuclei of Spodoptera frugiperda cells in culture was monitored at different times postinfection. Up to 8 h postinfection viral RNA synthesis remained sensitive to 5 mug of alpha-amanitin per ml. During the course of infection this sensitivity decreased, and at 24 h postinfection RNA synthesis was completely resistant to alpha-amanitin. DEAE-Sephadex profiles of RNA polymerase isolated at 24 h postinfection showed a new, chromatographically distinct, alpha-amanitin-resistant form whose kinetics and response to divalent cations differed from those of the host RNA polymerases. The possibility that this enzyme may be responsible for viral late transcription is discussed.

Journal Article↗
PubMed1989

Expression of Sindbis virus 26S cDNA in Spodoptera frugiperda (Sf9) cells, using a baculovirus expression vector.

To study protein processing in an insect Spodoptera frugiperda (fall armyworm; Sf9) cell line, a 26S cDNA encoding the sequence of Sindbis virus structural proteins (capsid protein, of 30 kilodaltons [kDa]; p62 [the precursor of E3 and E2], of 62 kDa; a 6-kDa peptide; and the E1 protein, of 56 kDa) was inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) adjacent to the polyhedrin promoter. By immunoblot analysis with antisera directed against whole Sindbis virus and the individual structural proteins (capsid, E2, and E1), we have shown that polypeptides similar in size and antigenicity to those synthesized in Sindbis virus-infected BHK cells are expressed in Sf9 cells infected with the recombinant baculovirus Ac373-SV26. By pulse-chase labeling in the presence or absence of tunicamycin, by endo-beta-N-acetylglucosaminidase H (endo-H) treatment of the recombinant glycoproteins, and by N-terminal sequence analysis of the E1 envelope glycoprotein, we have further shown that the 26S transcription translation unit of Sindbis virus, although normally encoded by nonnuclear RNA, is expressed and proteolytically cleaved similarly, if not identically, in Sf9 cells as compared with BHK cells when a baculovirus expression vector is used.

Animals↗
PubMed1991

Proteins encoded in the 81.2- to 85.0-map-unit fragment of Autographa californica nuclear polyhedrosis virus DNA can be translated in vitro and in Spodoptera frugiperda cells.

We have previously demonstrated that five open reading frames exist in the nucleotide sequence of the 81.2- to 85.0-map-unit (m.u.) segment of plaque isolate E of Autographa californica nuclear polyhedrosis virus (AcNPV) DNA. The corresponding polypeptides are 9.8, 12.1, 36.6, 25.0, and 48.2 kDa in size (C. Oellig, B. Happ, T. Müller, and W. Doerfler, J. Virol. 63:1494, 1989), and we have investigated whether these proteins can be translated in infected cells. On subfragments of this viral DNA segment, mRNAs were selected from AcNPV-infected Spodoptera frugiperda insect cells at different times postinfection (p.i.). The in vitro translation of these RNAs in a rabbit reticulocyte-derived cell-free translation system yielded polypeptides of approximately 10 to 11, 12 to 14, 28, 36 to 38, and 48 to 50-kDa which were commensurate in size with the theoretically expected values. mRNAs for the 28- and 48- to 50-kDa proteins were identified by their translation products at 6 h p.i., and mRNAs for the 10- to 11-, 12- to 14-, and 36- to 38-kDa proteins were identified by their translation products at 12 h p.i. We constructed an AcNPV recombinant which carried in its polyhedrin gene the 3.9-kbp EcoRI-HindIII (81.8 to 84.8 m.u.) subfragment of the EcoRI J segment. Nucleotide sequence determinations revealed that the intact polyhedrin promoter lay adjacent to the additional 81.8- to 84.8-m.u. fragment in this recombinant. In S. frugiperda cells, which were infected with the recombinant AcNPV, a protein of 36 to 38 kDa was detected at 44 h p.i. in larger amounts than after infection with the nonrecombinant virus. However, there was no evidence for larger amounts of RNA derived from the 81.8- to 84.8-m.u. fragment in recombinant-infected cells. Recombinant-infected cells lacked the polyhedrin polypeptide. The synthesis of the 36- to 38-kDa polypeptide in recombinant- or AcNPV-E-infected S. frugiperda cells could be demonstrated by immunoprecipitation experiments. Peculiarly, this polypeptide was present in the cytoplasm as a 64-kDa glycoprotein. These data corroborate the notion that at least some of the open reading frames encoded in the 81.2- to 85.0-m.u. segment of AcNPV can be expressed in S. frugiperda cells.

Amino Acid Sequence↗
PubMed1992

In vitro transactivation of baculovirus early genes by nuclear extracts from Autographa californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells.

Nuclear extracts, prepared from Autographa californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells during a time course of infection, were analyzed for activation of early gene transcription and for late gene transcription. The templates used in the in vitro transcription assays contained promoters for baculovirus genes that have been classified as immediate early, delayed early, and late. The promoters were derived from the baculovirus 39K, p26, gp64, and DNA polymerase genes. In addition, the adenovirus major late promoter was included in these studies. We found that transcription from promoters classified as immediate early or delayed early was accurately initiated by using extracts from uninfected cells. Furthermore, transcription from all early promoters tested was found to be transactivated by nuclear extracts prepared at 4 and 8 h postinfection. However, baculovirus enhancer-dependent transcriptional activation was not observed in tests with templates containing the hr5 enhancer sequence. Transcription from baculovirus late promoters was also not observed. A decline in transcription by nuclear extracts prepared from cells late in infection was associated with the presence of DNase activity.

Adenoviridae↗
PubMed1993

In vitro transcription from baculovirus late gene promoters: accurate mRNA initiation by nuclear extracts prepared from infected Spodoptera frugiperda cells.

Extracts prepared from nuclei of Autographa californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells were shown to support in vitro transcription from baculovirus late gene promoters. In vitro transcription was optimized for the late promoter of the 39K gene. The Mg2+ concentration was critical; concentrations higher than 1 to 2 mM did not support late transcription. Additional conditions included template (40 micrograms/ml), extract (2.5 mg/ml), and incubation time (25 min). Using a combination of runoff assays and high-resolution primer extension analyses, this system was shown to accurately initiate transcription from a variety of baculovirus late gene promoters, including those from the 39K and p39/capsid late genes and the hyperexpressed p10 and polyhedrin very late genes. In vitro transcription from the 39K late promoter was resistant to high concentrations of both alpha-amanitin (100 micrograms/ml) and tagetitoxin (4,000 U/ml), suggesting that neither RNA polymerase II nor III is responsible for the transcription of baculovirus late genes.

Animals↗
PubMed1985

DNA homology relationships between Spodoptera littoralis nuclear polyhedrosis virus and other baculoviruses.

The DNA sequence homology relationships between Spodoptera littoralis nuclear polyhedrosis virus (NPV) and five other lepidopteran NPVs were studied by hybridization of 32P-labeled NPV DNAs to Southern blots of restriction-endonuclease-digested NPV DNA. The S. littoralis NPV (SlMNPV) shows extensive DNA sequence homology through most of the genome with S. frugiperda NPV and S. exigua NPV. Heliothis armigera NPV, H. zea NPV, and Autographa californica NPV (AcMNPV) share fewer regions of homology with SlMNPV. Using the cloned HindIII fragment V of AcMNPV DNA, fragments with sequence homology to the polyhedrin gene of AcMNPV were identified for the viruses studied.

DNA Restriction Enzymes↗
PubMed1989

Expression of functional human acid beta-glucosidase in COS-1 and Spodoptera frugiperda cells.

A cDNA encoding human acid beta-glucosidase (N-acylsphingosyl-1-O-beta-D-glucoside: glucohydrolase, EC 3.2.1.45) expressed catalytically active enzyme in transfected COS-1 or infected Spodoptera frugiperda (Sf9) cells. The expression plasmid p91023(B) (p91023B/Glc) and a Baculovirus (AcMNPV/Glc) containing the cDNA were constructed and used with the respective cells. By immunoblotting a glycosylated, 63-kilodalton human acid-beta-glucosidase was detected in the transfected or infected cells. A 56-kilodalton human polypeptide was obtained after complete deglycosylation with N-Glycanase. The expressed human enzymes also had partial endoglycosidase H sensitivity. The human enzyme expressed at high levels in Sf9 cells and had normal immunologic properties. With the partially purified enzyme from Sf9 cells, intact function of active site was indicated by normal kcat and Kmapp or Kiapp values for alternative substrates or potent inhibitors, respectively. The expressed enzyme was also activated normally by the negatively charged lipid, taurocholate. The results of these studies indicate that the Baculovirus expression system could provide a convenient source of normal human enzyme for structure/function investigations. In addition, this expression system should prove useful for the identification and evaluation of putative etiologic point mutations in Gaucher disease variants with kinetically altered residual enzymes.

Animals↗
PubMed1990

Overexpression of full-length human glucocorticoid receptor in Spodoptera frugiperda cells using the baculovirus expression vector system.

We have expressed a full-length human glucocorticoid receptor (hGR) in Spodoptera frugiperda (Sf9) cells using the baculovirus expression vector system (BEVS). The level of expression is approximately 100-fold greater than in CEM-C7 cells. Between 0.5-1.0 mg hGR can be generated per liter of Sf9 cell culture. The expressed hGR is capable of binding glucocorticoids with specificity and high affinity. Covalent labeling with 3H-dexamethasone mesylate and Western blot analysis using a polyclonal antibody indicate that the molecular weight of the expressed protein is approximately 94 k. The nonactivated receptor sediments as a 8-9S complex in sucrose gradients and can be heat activated to a 4S form. The activated receptor is capable of retarding the migration of a 23 base-pair DNA fragment containing the glucocorticoid response element from the tyrosine aminotransferase gene. These data indicate that the expressed GR displays characteristics identical to those of GR from mammalian cells. By scaling up this culture we can, for the first time, obtain enough purified full-length receptor for crystallographic and functional studies which could provide new insight into exactly how hGR works.

Animals↗
PubMed1997

Behavioural analysis of olfactory conditioning in the moth spodoptera littoralis (Boisd.) (Lepidoptera: noctuidae)

We studied the associative learning capabilities for behaviourally relevant cues in the moth Spodoptera littoralis. The moths were trained to associate a conditioned stimulus (CS), geraniol odour, with an unconditioned stimulus (US), a sucrose solution. The occurrence of a proboscis extension reflex (PER) was tested. The PER performance during acquisition increased steadily with the number of training trials. Non-associative control procedures did not result in learning. PER conditioning was achieved when the CS was presented 1-3 s before the US. A wide range of inter-trial intervals was able to support conditioning. Males and females learned equally well. Moths could to some degree learn the CS-US association after a single trial. These results demonstrate that S. littoralis females and males have a good capability to associate an odour with a reward. The neural basis of olfactory coding in moths has been well studied; thus, the moth provides a powerful system in which to examine the neurobiology of olfactory learning.

Journal Article↗
PubMed1993

Isolation and identification of a cardioactive peptide from Tenebrio molitor and Spodoptera eridania.

We isolated several cardioactive peptides from extracts of whole heads of the mealworm, Tenebrio molitor, and the southern armyworm, Spodoptera eridania, using a semi-isolated heart of Manduca sexta for bioassay. We have now isolated from each species the peptide with the strongest effect on rate of contraction of the heart. The peptides were identified using micro Edman sequencing and mass spectrometric methods. This cardioactive peptide has the same primary structure from both species: Pro-Phe-Cys-Asn-Ala-Phe-Thr-Gly-Cys-NH2, a cyclic nonapeptide which is identical to crustacean cardioactive peptide (CCAP) originally isolated from the shore crab, Carcinus maenas, and subsequently isolated from Locusta migratoria and Manduca sexta. This is additional evidence that CCAP has widespread occurrence in arthropoda.

Amino Acid Sequence↗
PubMed2005

Behavior of Trichogramma atopovirilia Oatman & Platner and T. pretiosum Riley (Hymenoptera: Trichogrammatidae) on Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) egg masses.

In this study we analyzed the impact of physical barriers of Spodoptera frugiperda (J. E. Smith) egg-masses on the behavior of Trichogramma atopovirilia Oatman & Platner and Trichogramma pretiosum Riley. The duration of drumming, drilling, oviposition, period spent over the egg-mass, and interval between parasitized eggs were timed, and the number of parasitized eggs were recorded. The presence of scales on the egg-masses caused a significant increase in the time spent by both parasitoids on each process and a decrease in the residence time over the egg-mass and in the number of parasitized eggs, with an increase in the number of egg layers. There was a significant decrease in the number of parasitized eggs in relation to egg-masses with one layer and no scales. We observed that the physical barriers in fall armyworm egg-masses changed the behavior of T. atopovirilia and T. pretiosum, affecting their parasitization capacity.

Animals↗
PubMed2001

An insensitive acetylcholinesterase confers resistance to methomyl in the beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae).

Two forms of acetylcholinesterase were identified in field populations of the beet armyworm, Spodoptera exigua (Hübner), collected from cotton in San Joaquin Valley, CA. Strains (BESS and BKRR) homogeneous for each variant were isolated and their relative susceptibilities to methomyl, chlorpyrifos, and chlorpyrifos-oxon assessed by topical application bioassay. In comparisons with a laboratory susceptible strain (DOW), BKRR and BESS expressed 68-fold and sevenfold resistance, respectively, to the carbamate methomyl. Neither strain was cross-resistant to chlorpyrifos or its oxygen analog (chlorpyrifos-oxon). In biochemical studies, the BKRR AChE enzyme was approximately 30-fold and sixfold more insensitive to methomyl and chlorpyrifos-oxon, respectively, compared with the DOW enzyme. The correlation between the toxicological and biochemical studies provides strong evidence that target-site insensitivity is an important mechanism of resistance to methomyl. The lack of significant cross-resistance to chlorpyrifos suggests that the insensitive AChE in these field populations was selected by methomyl alone and not by the organophosphate.

Acetylcholinesterase↗
PubMed2006

Molecular variability of Spodoptera frugiperda (Lepidoptera: Noctuidae) populations associated to maize and cotton crops in Brazil.

The molecular variability among 10 populations of Spodoptera frugiperda (J.E. Smith), collected from maize, Zea mays L., or cotton Gossypium hirsutum L. crops located at distinctive geographical regions in Brazil, was assessed through random amplification of polymorphic DNA (RAPD)-polymerase chain reaction (PCR). In total, 208 RAPD markers were evaluated, and 98% of them were polymorphic. The mean genetic similarity was 0.6621 and 0.2499 by the Simple Matching and Jaccard matrices, respectively. In general, the unweighted pair-group method with arithmetic average dendrograms separated the populations into clusters related to the geographical origin of the samples. No branch of the dendrograms underpinning a molecular association of S. frugiperda has been identified to either of the two host plants. The molecular variance analysis showed that 18 and 82% of the genetic variation was distributed among and within the groups of populations, respectively. The principal coordinate analysis reinforced the pattern of population clustering found with the unweighted pair-group method with arithmetic average method. These results suggest the occurrence of considerable gene flow between S. frugiperda populations from maize and cotton fields located in the same region in Brazil. Therefore, for an effective management of this pest, there is an urgent need for a better understanding of the gene flow of S. frugiperda populations associated to different host plants along the distribution range of this pest over time in a specific cropping system.

Animals↗
PubMed2006

Importance of host plant species, Neotyphodium endophyte isolate, and alkaloids on feeding by Spodoptera frugiperda (Lepidoptera: Noctuidae) larvae.

Three grass host species--tall fescue, Festuca arundinacea Schreber; meadow fescue, Festuca pratensis Hudson; and perennial ryegrass, Lolium perenne L.--each infected with a number of different Neotyphodium endophyte isolates, were investigated for their effects on fall armyworm, Spodoptera frugiperda (J.E. Smith). Alkaloid profiles varied among associations. Choice and no-choice tests comparing feeding and early development of S. frugiperda larvae on endophyte-infected and endophyte-free leaf blade material were performed. Endophyte-mediated resistance to S. frugiperda was greatest in meadow fescue and weakest in tall fescue. Some endophyte isolates, particularly in perennial ryegrass and meadow fescue, had a major effect on feeding and development of S. frugiperda, whereas others had no effect or were only weakly efficacious. In tall fescue, some associations deterred S. frugiperda from feeding in choice tests but had no effect on development, whereas larvae reared on other associations weighed significantly more than control larvae fed endophyte-free grass. It was concluded that the deleterious consequences of endophyte infection were easily masked by other factors in tall fescue. Relative leaf age had no effect on feeding preferences in the three host species. Chemical analysis of herbage from the plants used, and results from a no-choice study using spiked artificial diets, failed to individually implicate any of the major known alkaloids (peramine, lolitrem B, ergovaline, and lolines) in the observed effects on S. frugiperda. Hypotheses explaining these observations, and their impact on creating desirable grass-endophyte associations for use in pastures, are discussed.

Alkaloids↗
PubMed2004

Flight capability and fatty acid level in triacylglycerol of long-distance migratory adults of the common cutworm, Spodoptera litura.

The larvae of Spodoptera litura were reared on an artificial diet, and the flight capability, and triacylglycerol (TG) level plus its fatty acid composition in 3-day-old sexually mature and non-fed adults were compared. In males, during 3 hr of tethered flight, the levels of abdominal TG and its fatty acid components did not change. But thereafter the TG and fatty acids, significantly unsaturated fatty acids in TG declined in their levels with the prolongation of flight, unsaturated fatty acids being exhausted preceding saturated fatty acid decline. When males were tested by tethered flight for 20 hr, some could fly for nearly the whole period, and were judged to be able to fly for approximately 24 hr, depending on the level of residual TG. Fatty aids in TG decreased in females similarly to males during tethered flight and some females with fully developed ovaries deposited eggs after 12 hr of flight similarly to non-flown individuals, which supports the long-distance flight capability even in sexually mature females. These results are discussed with regard to the overseas migration of this moth.

Animal Migration↗