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PubMed2003

Lysozyme of the beet armyworm, Spodoptera exigua: activity induction and cDNA structure.

Lysozyme of the beet armyworm, Spodoptera exigua, was characterized in its up-regulation pattern, and its cDNA was cloned by RT-PCR using degenerate primers designed from some conserved amino acid regions shared with related lepidopteran species. Lysozyme activity of the non-immunized S. exigua had developmental variation, with the highest level in the fifth instar larvae. The basal level of the lysozyme activity was significantly enhanced by the injection of laminarin or lipopolysaccharide (LPS). Among different LPSs tested, the extract from an entomopathogenic bacterium, Xenorhabdus nematophilus, proved to be the most potent. Fat body was the major tissue to express the lysozyme in S. exigua. Even though there was a significantly elevated level of lysozyme in the hemolymph at 12 h after laminarin injection, the transcript ( approximately 1.1 kbp) was found in the fat body as early as 6 h after injection. The cDNA of the lysozyme was cloned as 602 bp with a deduced 141-amino-acid residue open reading frame containing two introns. Except for a signal peptide with 20 amino acid residues, the estimated molecular weight and isoelectric point of the lysozyme was 14313.83 Da and 8.59, respectively. Only a single copy gene of the lysozyme was found in S. exigua genome from Southern analysis. The amino acid sequence of S. exigua lysozyme showed higher similarity (88.7%) with noctuid species compared to other lepidopteran species.

Amino Acid Sequence↗
PubMed1998

Expression of bovine activin-A and inhibin-A in recombinant baculovirus-infected Spodoptera frugiperda Sf 21 insect cells.

Currently, bioactive activin and inhibin for investigative purposes are obtained either by purification from bovine or porcine follicular fluid or have been kindly supplied in limited amounts by Genentech. The latter are recombinant formulations produced in cultured monkey kidney CV-1 cells. The aims of this study were to assess the potential of the baculovirus expression system as an alternative means to produce recombinant activin and inhibin. Towards these goals, two recombinant baculoviruses, AcBovACTA and AcBovINHA, were constructed. AcBovACTA contains a contiguous copy of the bovine beta A-inhibin/activin structural gene encoding the beta A-preproprotein whereas AcBovINHA contains contiguous copies of the bovine alpha-inhibin and beta A-inhibin/activin structural genes encoding the alpha- and beta A-preproproteins, respectively. Western blot analyses, using monoclonal antibodies specific for the mature portions of the alpha-inhibin and beta A-inhibin/activin subunits, demonstrated that Spodoptera frugiperda Sf21 cells infected with either recombinant virus secreted mature homodimeric activin-A into the medium. In addition, Sf21 cells infected with the recombinant AcBovINHA virus were found also to produce substantial amounts of the alpha-inhibin precursor protein. However, the mature portion of the latter is not secreted into the medium but is retained within infected cells in an incompletely processed form(s). The recombinant activin-A secreted by Sf21 cells infected with the AcBovACTA virus was shown to possess activin bioactivity when analysed by in vitor bioassay and, therefore, provides an alternative route to mammalian cell expression for the production of recombinant activin-A.

Activins↗
PubMed2006

Life history of Spodoptera exigua (Lepidoptera: Noctuidae) on various host plants.

The incubation period of Spodoptera exigua (Hübner) was not influenced by the host plant, whereas larval development time and pupal period were affected. Larval development time was longest on shallot and lady's finger, followed by cabbage and long bean. Larvae did not develop beyond the first instar when fed on chilli. The pupal period was longer on lady's finger than on cabbage, shallot and long bean. Overall, adult longevity was not influenced by the host plant but there was a difference between female and male longevity among the host plants. Survival of S. exigua was affected by the host plant at the larval stage. The number of larval instars varied between 5 and 8 within and between the studied host plants. Long bean was found to be the most suitable host plant and provide the best food quality for S. exigua compared to the other host plants, as it allowed faster development, fewer larval instars and a higher survival rate.

Abelmoschus↗
PubMedDate not supplied

Expression of oligohistidine-tagged ricin B chain in Spodoptera frugiperda.

DNA encoding ADPGH6G was fused to the 5'-end of RTB DNA and subcloned as a BamHI-EcoRI DNA cassette into the baculovirus transfer vector, pAcGP67A. Spodoptera frugiperda Sf9 cells were cotransfected with pAcGP67A-ADPGH6G-RTB DNA and BaculoGold AcNPV DNA, and recombinant baculovirus was isolated by two cycles of limiting dilution assay followed by dot blot analysis with 32P-dCTP random primer labeled RTB DNA. Recombinant virus was purified and amplified to obtain stocks at titers of 10(7) infectious particles/mL. Sf9 cells grown in serum-free medium were then infected at an moi of 3 in the presence of 25 mM alpha-lactose. After 5 days, supernatants and cell pellets were harvested and assayed by an asialofetuin ELISA for recombinant RTB protein. Fusion RTB protein was produced in the supernatant at 5 mg/L and in the cell pellet at 1 mg/L. Recombinant protein was purified to > 80% homogeneity using either a monoclonal antibody affinity matrix with alkaline elution or a Ni(2+)-NTA matrix with imidazole elution. The purified protein bound asialofetuin similarly to plant RTB. N-terminal sequencing confirmed the oligohistidine tag. SDS-PAGE confirmed the 1,000 Da increase in mass relative to "wild-type" recombinant RTB produced in Sf9 cells. Immunoblots confirmed reactivity with polyclonal and monoclonal antibodies to plant RTB. The fusion protein reassociated with plant RTA similarly to plant RTB.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2003

Stress-induced apoptosis in Spodoptera frugiperda (Sf9) cells: baculovirus p35 mitigates eIF2 alpha phosphorylation.

Spodoptera frugiperda (Sf9) ovarian cells, natural hosts for baculovirus, are good model systems to study apoptosis and also heterologous gene expression. We report that uninfected Sf9 cells readily undergo apoptosis and show increased phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) in the presence of agents such as UVB light, etoposide, high concentrations of cycloheximide, and EGTA. In contrast, tunicamycin, A23187, and low concentrations of cycloheximide promoted eIF2alpha phosphorylation in Sf9 cells but without apoptosis. These findings therefore suggest that increased eIF2alpha phosphorylation does not always necessarily lead to apoptosis, but it is a characteristic hallmark of stressed cells and also of cells undergoing apoptosis. Cell death induced by the above agents was abrogated by infection of Sf9 cells with wild-type (wt) AcNPV. In contrast, Sf9 cells when infected with vAcdelta35, a virus carrying deletion of the antiapoptotic p35 gene, showed increased apoptosis and enhanced eIF2alpha phosphorylation. Further, a recombinant wt virus vAcS51D expressing human S51D, a phosphomimetic form of eIF2alpha, induced apoptosis in UVB pretreated Sf9 cells. However, infection with vAcS51A expressing a nonphosphorylatable form (S51A) of human eIF2alpha partially reduced apoptosis. Consistent with these findings, it has been observed here that caspase activation has led to increased eIF2alpha phosphorylation, while caspase inhibition by z-VAD-fmk reduced eIF2alpha phosphorylation selectively in cells exposed to proapoptotic agents. These findings therefore suggest that the stress signaling pathway determines apoptosis, and caspase activation is a prerequisite for increased eIF2alpha phosphorylation in Sf9 cells undergoing apoptosis. The findings also reinforce the conclusion for the first time that the "pancaspase inhibitor" baculovirus p35 mitigates eIF2alpha phosphorylation.

Animals↗
PubMed1999

Cryptoregiochemistry of the delta11-myristoyl-CoA desaturase involved in the biosynthesis of Spodoptera littoralis sex pheromone.

Many moth species biosynthesize their sex pheromones by the action of unique desaturases. These membrane-bound family of enzymes are especially interesting, since some of them produce (E)-unsaturated fatty acids either exclusively or along with the (Z)-isomer. In this article we present the first mechanistic study on one of these enzymes, namely, the Delta11-myristoyl-CoA desaturase of the moth Spodoptera littoralis. Intermolecular primary isotope effect determinations were performed in competition experiments. The unusual use of odd-number fatty acids, tridecanoic acid and deuterium-labeled tridecanoic acid, in these experiments showed the existence of a large isotope effect for the carbon-hydrogen bond cleavage at C11, but no isotope discrimination occurred in the removal of C12-H. The results of the competitive experiments are consistent with the hypothesis that this Delta11-desaturase involves a first slow, isotope-sensitive C11-H bond cleavage, with probable formation of an unstable intermediate, followed by a second fast C12-H bond removal. We suggest that a single enzyme may be responsible for the formation of both (Z)- and (E)-11-tetradecenoic acids by accommodating both gauche and anti conformers of the substrate, respectively. It is also possible that two mechanistically identical discrete enzymes are involved in each desaturation. In this case, the geometry of the resulting double bond would result from the different conformation adopted by the acyl substrate at each enzyme active site.

Animals↗
PubMedDate not supplied

Chromosome instability in Spodoptera frugiperda Sf-9 cell line.

Homogeneous cell lines are essential in industry and research if reliable and reproducible data are to be obtained. The Spodoptera frugiperda (Sf-9) cell line routinely used for the production of recombinant proteins was found to be heterogeneous, containing a mixture of diploid and tetraploid cells. Using dilution-cloning techniques, diploid and tetraploid subpopulations were isolated from a Sf-9 parental cell line, and their cytogenetic state was monitored using Vinblastine to arrest cells in mitosis. Flow cytometry was used to obtain a snapshot of the predominant subpopulations present to verify the karyological results. The rate at which clonal populations digress into the heterogeneous state was found to be more rapid for the diploid subpopulation, with the emergence of tetraploid cells after only 11 passages, than for the tetraploid subpopulation, where diploid clones appeared after 18 passages. The chromosomes in both diploid and tetraploid subpopulations as well as the parental cell line were found to spontaneously fragment during growth and expansion processes, giving rise to variable chromosome numbers. DNA analysis of cell lines obtained from laboratories worldwide have shown that the Sf-9 cell line used for the production of many recombinant proteins is cytologically unstable, leading to varying degrees of polyploidal state depending on its culture history and supplier.

Animals↗
PubMedDate not supplied

Insights into the central metabolism of Spodoptera frugiperda (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5) insect cells by radiolabeling studies.

The insect cell baculovirus expression vector system (BEVS) is one of the most commonly used expression systems for recombinant protein production. This system is also widely used for the production of recombinant virus and virus-like particles. Although several published reports exist on recombinant protein expression using insect cells, information dealing with their metabolism in vitro is relatively scarce. In this work we have analyzed the metabolism of glucose and glutamine, the main carbon and/or energy compounds, of the two most commonly used insect cell lines, Spodoptera frugiperda (Sf-9) and the Trichoplusia ni BTI-Tn-5B1-4 (Tn-5). Radiolabeled substrates have been used to determine the flux of glucose carbon entering the tricarboxylic acid cycle (TCA) and the pentose phosphate (PP) pathway by direct measurement of 14CO2 produced. The percentage of total glucose metabolized to CO2 via the TCA cycle was higher in the case of the Sf-9 (2.7%) compared to Tn-5 (0.6%) cells, while the percentage of glucose that is metabolized via the PP pathway was comparable at 14% and 16% for the two cell lines, respectively. For both cell lines, the remaining 83% of glucose is metabolized through other pathways generating, for example, lactate, alanine, etc. The percentage of glutamine oxidized in the TCA cycle was approximately 5-fold higher in the case of the Tn-5 (26.1%) as compared to the Sf-9 cells (4.6%). Furthermore, the changes in the metabolic fluxes of glucose and glutamine in Tn-5-PYC cells, which have been engineered to express a cytosolic pyruvate carboxylase, have been studied and compared to the unmodified cells Tn-5. As a result of this metabolic engineering, significant increase in the percentage of glucose oxidized in the TCA cycle (3.2%) as well as in the flux through the PP pathway (34%) of the Tn-5-PYC were observed.

Animals↗
PubMed2001

Acute, sublethal, antifeedant, and synergistic effects of monoterpenoid essential oil compounds on the tobacco cutworm, Spodoptera litura (Lep., Noctuidae).

Monoterpenoids (terpenes and biogenically related phenols) commonly found in plant essential oils were tested for acute toxicity via topical application to tobacco cutworms (Spodoptera litura Fab.). The most toxic among 10 such compounds was thymol (LD(50) = 25.4 microg/larva) from garden thyme, Thymus vulgaris. The compounds were then tested for sublethal effects, specifically inhibition of larval growth after topical application of low doses. Among 6 compounds tested, an LD(10) dose reduced growth by 20% on average 3 days after administration. Feeding deterrence was determined using a cabbage leaf disk choice test. The most deterrent compound was thymol, with a DC(50) of 85.6 microg/cm(2) leaf disk area. Because minor constituents in complex essential oils have been suggested to act as synergists, binary mixtures of the compounds were tested for synergy vis à vis acute toxicity and feeding deterrence. trans-Anethole acted synergistically with thymol, citronellal, and alpha-terpineol, in terms of both acute toxicity and feeding deterrence. On the basis of these findings, several complex mixtures were developed and tested as leads for effective control agents. Candidate mixtures demonstrated good synergistic effects. The observed LD(50) of mixture 3 was 40.6 microg/larvae compared to an expected value of 74.6 microg/larvae. The result of this research is a proprietary product suitable for commercial production.

Animals↗
PubMed2001

Biotransformation of (R)- and (S)-terpinen-4-ol by the larvae of common cutworm (Spodoptera litura).

(R)-Terpinen-4-ol was mixed in an artificial diet at a concentration of 1 mg/g of diet, and the diet was fed to the last instar larvae of common cutworm (Spodoptera litura). Metabolites were recovered from frass and analyzed spectroscopically. (R)-Terpinen-4-ol was transformed mainly to (R)-p-menth-1-en-4,7-diol. Similarly, (S)-terpinen-4-ol was transformed mainly to (S)-p-menth-1-en-4,7-diol. The C-7 position (allylic methyl group) of (R)- and (S)-terpinen-4-ol was preferentially oxidized.

Animals↗
PubMed2002

Activity of octylthiotrifluoropropan-2-one, a potent esterase inhibitor, on growth, development, and intraspecific communication in Spodoptera littoralis and Sesamia nonagrioides.

A series of experiments were conducted to examine the effect of 3-octylthio-1,1,1-trifluoro-2-propanone (OTFP) on growth, development, and behavior of the cotton leafworm, Spodoptera littoralis (Lepidoptera: Noctuidae), and the corn stalk borer, Sesamia nonagrioides (Lepidoptera: Noctuidae). The chemical behaved as an oviposition deterrent and, when added to the diet of the second-instar larvae of both insects, reduced diet consumption and growth, pupation, and adult emergence. Treatment of 100-5000 ng of the compound on fourth-instar larvae for 3-24 h, however, did not produce significant differences in the amount of diet ingested. Our results suggest that the effect of OTFP is long-lasting and that the inhibitor is not fully detoxified by the detoxification enzymes of the digestive tract of the insects. In behavioral assays, adult males which had been treated with the chemical at the larval stage were less attracted to the pheromone source than regular untreated males. When Sp. littoralis untreated females were used as the attractant source, treated males also displayed significantly fewer contacts with the cage-containing females than untreated or solvent-treated males. In the presence of treated females, only 27% of treated males successfully completed the flight in comparison to animals responding to solvent-treated females (54.5%). By contrast, when Se. nonagrioides females, whether they had been subjected or not to the treatment, were used as the attractant source, males were similarly attracted to them regardless of whether they had been treated or not at the larval stage. Analyses of gland extracts of Sp. littoralis treated females showed no difference from control insects in the qualitative or quantitative composition of the pheromone blend. The results obtained, in combination with other results previously reported by us (Riba, M.; Sans, A.; Bau, P.; Grolleau, G.; Renou, M.; Guerrero, A. J. Chem. Ecol. 2001, 27, 1879-1897), provide new and relevant information about the possible utility of these chemicals in future studies directed to the development of new approaches for pest control.

Acetone↗
PubMed2002

Biotransformation of alpha-terpineol by the larvae of common cutworm (Spodoptera litura).

Biotransformation of alpha-terpineol by the common cutworm (Spodoptera litura) larvae was investigated. alpha-Terpineol was mixed in an artificial diet, and the diet was fed to the larvae (fourth-fifth instar) of S. litura. Metabolites were isolated from the frass and analyzed spectroscopically. Main metabolites were 7-hydroxy-alpha-terpineol (p-menth-1-ene-7,8-diol) and oleuropeic acid (8-hydroxy-p-menth-1-en-7-oic acid). Intestinal bacteria from the frass of larvae did not participate in the metabolism of alpha-terpineol. alpha-Terpineol was preferentially oxidized at the C-7 position (allylic methyl group) by S. litura larvae.

Animals↗
PubMed2002

Citrus limonoids and their semisynthetic derivatives as antifeedant agents against Spodoptera frugiperda larvae. A structure-activity relationship study.

The antifeedant activity of Citrus-derived limonoids limonin (1), nomilin (2), and obacunone (3) and their semisynthetic derivatives 4-26 was evaluated against a commercially important pest, Spodoptera frugiperda. Simple chemical conversions were carried out on the natural limonoids obtained from seeds of Citrus limon. These conversions focused on functional groups considered to be important for the biological activity, namely the C-7 carbonyl and the furan ring. In particular, reduction at C-7 afforded the related alcohols, and from these their acetates, oximes, and methoximes were prepared. Hydrogenation of the furan ring was also performed on limonin and obacunone. The known antifeedant properties of the Citrus limonoids are confirmed. Comparison with previously reported data shows that insect species vary in their behavioral responses to these structural modifications. Highly significant antifeedant activity (P < 0.01) for two natural (1 and 3) and three semisynthetic limonoids (4, 8, and 10) was observed against S. frugiperda.

Animals↗
PubMed2003

Insect antifeedant activity of flavones and chromones against Spodoptera litura.

The antifeedant polymethylated flavones 5-hydroxy-3,6,7,8,4'-heptamethoxyflavone, 5-hydroxy-3,6,7,8-tetramethoxyflavone, and 5,6-dihydroxy-3,7-dimethoxyflavone have been isolated from the cudweed, Gnaphalium affine D. Don (Compositae). These flavonoids and authentic analogues showed insect antifeedant activity against the common cutworm (Spodoptera litura F.). In a previous paper, it was suggested that there was no substituent on the B-ring of the flavonoid for the beneficial antifeedant activity against the common cutworm. These flavonoids having a phenyl group as the B-ring and the chromone as elimination of the B-ring from the flavonoids were used to test the hypothesis of the previously described B-ring effect. The known fact is that Sculletaria baicarensis (Rutaceae) produced the 2-phenyl flavone. Test compounds and their methylated derivatives were prepared from this material for the structure-activity relationship (SAR) study of insect antifeedant activity. In spite of the 2-phenyl flavonoids, some tested compounds did not show any insect antifeedant activity against the common cutworm, although these inactive flavonoids were deficient in the 6-substituent group on the A-ring of the flavonoid. This 6-position-substituted derivative almost showed strong insect antifeedant activity against common cutworm. Moreover, the tested flavonoids having a hydroxyl group as a substituent on any of the positions tended to increase the activity. These results suggested the importance of the 6-position substitution on the flavonoid; however, hydrophilic substituents decreased the activity. Baicalein (5,6,7-trihydroxyflavone) derivatives did not show any activity despite having the 6-substituent derivative. Although the activity of some chromones increased the activity of the flavone, the bulky B-ring was a disadvantage for the antifeedant activity. It was suggested that the charge on C(3) and C(5) of the flavonoid was important for the biological activity. Additionally, an adequate hydrogen bonding property, which is different from lipophilicity, was an advantage for the activity on the basis of a QSAR analysis.

Animals↗
PubMed2003

Antifeedant and growth inhibitory effects of some neo-clerodane diterpenoids isolated from Clerodendron species (Verbenaceae) on Earias vitella and Spodoptera litura.

Antifeedant and growth inhibitory effects of various neo-clerodane diterpenoids having a furofuran moiety, isolated from Clerodendron spp., were studied using Earias vitella and Spodoptera litura. The compounds clerodendrin B, 3-epicaryoptin, 15-hydroxyepicaryoptin, and clerodin were effective antifeedants at 10 microg/cm(3) (30 microg/g) of diet against E. vitella and at 10 microg/cm(2) of leaf against S. litura. All of the tested compounds, namely, clerodendrin B, 3-epicaryoptin, clerodendrin C, 15-hydroxyepicaryoptin, clerodendrin B acetate, and clerodin, showed good insect growth inhibitory activity even at lower concentrations.

Animals↗
PubMed2004

Metabolism of thymol and trans-anethole in larvae of Spodoptera litura and Trichoplusia ni (Lepidoptera: Noctuidae).

Metabolism of the monoterpenoid thymol and the phenylpropanoid trans-anethole, both constituents of essential oils, was investigated following topical and oral administration of the compounds to the tobacco cutworm (Spodoptera litura) and the cabbage looper (Trichoplusia ni). In both species and irrespective of route, administration of thymol resulted in the excretion of its 3-O-beta-glucoside, whereas trans-anethole was hydroxylated on the side chain methyl group. Both metabolites were isolated and their structures elucidated by interpretation of their mass and NMR spectra. Previous experiments indicated that trans-anethole synergized the toxicity of thymol in S. litura, but analyses of feces indicated that metabolism of thymol was not significantly suppressed when the two compounds were orally coadministered to T. ni larvae.

Allylbenzene Derivatives↗
PubMed2004

Elevated atmospheric CO(2) affects the chemical quality of brassica plants and the growth rate of the specialist, Plutella xylostella, but not the generalist, Spodoptera littoralis.

Cabbage, Brassica oleracea subsp. capitata (cv. Lennox and Rinda), and oilseed rape, Brassica rapa subsp. oleifera (cv. Valo and Tuli), plants were grown under ambient CO(2) (360 ppm) or elevated CO(2) (720 ppm) at 23/18 degrees C and under a photoperiod of 22/2 h light (250 micromol m(-)(2) s(-)(1))/dark regime for up to 5 weeks. Afterward, the performance of the crucifer specialist Plutella xylostella (Lepidoptera: Plutellidae) and the generalist Spodoptera littoralis (Lepidoptera: Noctuidae) on those plants was studied. The mean relative growth rate (RGR) of P. xylostella larvae, feeding on both cultivars of oilseed rape or on the Lennox cultivar of cabbage leaves grown at an elevated CO(2) concentration, was significantly reduced as compared to ambient CO(2). A negative larval growth rate at elevated CO(2) was observed for P. xylostella on both oilseed rape cultivars, but the growth rate was reduced but positive on cabbage. Conversely, the RGR of S. littoralis on either plant species was not affected by CO(2) treatment but was lower on cabbage cv. Rinda than on cv. Lennox. The mortality of the larvae was not affected by CO(2) treatment either. At the same time, elevated CO(2) significantly decreased the concentrations of leaf phytochemical constituents in oilseed rape, i.e., total phenolics and total nitrogen, but not in cabbage. The effect of elevated CO(2) on the leaf glucosinolate concentrations of both plant species was marginal. In addition, the observed significant changes in individual glucosinolate concentrations of oilseed rape leaves were not consistent among cultivars. However, our results demonstrate for the first time quite strong effects of CO(2) enrichment on the larval performance of P. xylostella, which is an important pest of Brassica plants around the world. Further studies are still required to increase our understanding of why elevated CO(2) differently affects the performance of specialist and generalist insect herbivores on Brassica plants.

Animals↗
PubMed1999

Biotransformation of (+)- and (-)-menthol by the larvae of common cutworm (Spodoptera litura).

(+)-Menthol was mixed in an artificial diet at a concentration of 1 mg/g of diet, and the diet was fed to the last instar larvae of common cutworm (Spodoptera litura). Metabolites were recovered from frass and analyzed spectroscopically. (+)-Menthol was transformed mainly to (+)-7-hydroxymenthol. Similarly, (-)-menthol was transformed mainly to (-)-7-hydroxymenthol. The C-7 position of (+)- and (-)-menthol was preferentially oxidized.

Animals↗