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PubMed2005

Insecticidal activity of some essential oils against larvae of Spodoptera littoralis.

Thirty-four essential oils were tested for insecticidal activity (fumigation or topical application) against larvae Spodoptera littoralis. Twenty essential oils applied by fumigation were highly toxic to the third instar of S. littoralis larvae. Two essential oils Nepeta cataria and Thuja occidentalis were highly toxic, with LC50< or =10.0 ml/m3. Five essential oils Salvia sclarea, Thymus mastichina, Origanum majorana, Pogostemon cablin and Mentha pulegium were toxic with LC50 between 10.1 and 20.0 l ml/m3. Twenty-three essential oils were highly toxic to the third instar of S. littoralis larvae after topical application. Eight essential oils Mentha citrata, N. cataria, S. sclarea, O. vulgare, O. compactum, Melissa officinalis, T. mastichina, and Lavandula angustifolia were highly toxic with LD50 < or =0.05 microl/larvae.

Administration, Topical↗
PubMed2006

Characterization of two homologues of ChaB in Spodoptera litura multicapsid nucleopolyhedrovirus.

ChaB, a putative regulator of ChaA in Escherichia coli (E. coli), and its homologues constitute a multigene family known to occur among bacteria, archaeabacteria and baculoviruses. Distinguished from E. coli ChaB, baculoviruses ChaB proteins lack a charged loop that can bind to Ca2+ and Mg2+. The baculovirus Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) contains two homologues of ChaB, open reading frames (ORFs) 52 (Sl52) and 53 (Sl53). Reverse transcription-PCR and 5' Race analyses indicated that transcription of both SpltMNPV chaB genes occurs by 12 h postinfection and is initiated at a late consensus (A/T)TAAG motif. Immunoblot analysis showed that Sl52 was expressed as a doublet of 23 and 26 kDa in infected S. litura cells, while Sl53 was expressed as a 16 kDa protein. Biochemical fractionation analysis indicated that the 23 kDa form of Sl52 was distributed in both cytoplasm and nucleus of infected cells, whereas the 26 kDa form of Sl52 and Sl53 proteins were only present in nucleus. Further analysis revealed that these proteins are associated with the nucleocapsid of occlusion-derived virus. Using a histone extraction protocol, the Sl53 and 26 kDa form of Sl52 proteins were both detected in the histone H1 fraction. Additionally, column chromatography analysis showed that the Sl52 and Sl53 proteins could interact with nucleic acids. It was proposed that SpltMNPV ChaB might function as DNA binding proteins.

Amino Acid Sequence↗
PubMed2004

Inhibition of the acyl-CoA desaturases involved in the biosynthesis of Spodoptera littoralis sex pheromone by analogs of 10,11-methylene-10-tetradecenoic acid.

The desaturase inhibitory activity of the cyclopropenyl alcohols 9,10-methylene-9-tetradecen-1-ol (9-MTOL), 10,11-methylene-10-tetradecen-1-ol (10-MTOL) and 11,12-methylene-11-tetradecen-1-ol (11-MTOL), which are structural analogs of 10,11-methylene-10-tetradecenoic acid (10-MTA), is reported. At equimolar ratios with respect to the different substrates, the three compounds completely inhibited the three desaturation reactions involved in the biosynthesis of Spodoptera littoralis sex pheromone. The dose-dependence of inhibition was determined for 10-MTA and its alcohol derivative. Both compounds inhibited the transformation of perdeuterated palmitic acid into perdeuterated (Z)-11-hexadecenoic acid and that of (E)-11-tridecenoic acid into (Z,E)-9,11-tridecadienoic acid with similar IC(50) values. The overall results presented in this work support scattered data that neither the free carboxyl groups nor their acyl-CoA esters are a requisite for inhibition of desaturases. Since the synthesis of cyclopropenols is much more convenient than that of cyclopropene fatty acids, this finding is of economical relevance regarding the putative use of cyclopropene derivatives in pest control.

Animals↗
PubMed2004

A genomic BAC library and a new BAC-GFP vector to study the holocentric pest Spodoptera frugiperda.

Two genomic tools for the study of Lepidoptera and the holocentric structure of their chromosomes are presented in this paper. A bacterial artificial chromosome (BAC) library was constructed using nuclear DNA partially digested with HindIII from eggs of Spodoptera frugiperda. The library contains a total of 36,864 clones with an average insert size of 125 kb, which corresponds to approximately 11.5 genome equivalents. Hybridization screening of the library was performed with eight single-copy genes, giving an average hit of 10 clones per marker gene. Colinearity between the genome and BACs was demonstrated at the triose phosphate isomerase (tpi) locus. Probing of the library with a PCR fragment internal to the 18S ribosomal gene allowed an estimation of the rDNA locus size close to 115 repeats per haploid genome. A new vector (pBAC3.6eGFP) for transient transfection into S. frugiperda cell lines has been constructed. It is based on the BAC vector, pBAC3.6e, in which a gene encoding GFP was inserted under the control of the densovirus P9 promoter. This vector has the advantage to accommodate large genomic inserts and to be transfected in a large lepidopteran host range. It was used to construct a second BAC library from Sf9 cell nuclear DNA in order to allow a comparison between somatic and cell line genome organization.

Animals↗
PubMed2004

Expression and evolution of delta9 and delta11 desaturase genes in the moth Spodoptera littoralis.

Desaturation of fatty acids is a key reaction in the biosynthesis of moth sex pheromones. The main component of Spodoptera littoralis sex pheromone blend is produced by the action of Delta11 and Delta9 desaturases. In this article, we report on the cloning of four desaturase-like genes in this species: one from the fat body (Sls-FL1) and three (Sls-FL2, Sls-FL3 and Sls-FL4) from the pheromone gland. By means of a computational/phylogenetic method, as well as functional assays, the desaturase gene products have been characterized. The fat body gene expressed a Delta9 desaturase that produced (Z)-9-hexadecenoic and (Z)-9-octadecenoic acids in a (1:4.5) ratio, whereas the pheromone gland Sls-FL2 expressed a Delta9 desaturase that produced (Z)-9-hexadecenoic and (Z)-9-octadecenoic acids in a (1.5:1) ratio. Although both Delta9 desaturases produced (Z)-9-tetradecenoic acid from myristic acid, transformed yeast grown in the presence of a mixture of myristic and (E)-11-tetradecenoic acids produced (Z,E)-9,11-tetradecadienoic acid, but not (Z)-9-tetradecenoic acid. The Sls-FL3 gene expressed a protein that produced a mixture of (E)-11-tetradecenoic, (Z)-11-tetradecenoic, (Z)-11-hexadecenoic and (Z)-11-octadecenoic acids in a 5:4:60:31 ratio. Despite having all the characteristics of a desaturase gene, no function could be found for Sls-FL4.

Amino Acid Sequence↗
PubMed2005

Identification and recombinant expression of a novel chymotrypsin from Spodoptera exigua.

A novel chymotrypsin which is expressed in the midgut of the lepidopteran insect Spodoptera exigua is described. This enzyme, referred to as SeCT34, represents a novel class of chymotrypsins. Its amino-acid sequence shares common features of gut chymotrpysins, but can be clearly distinguished from other serine proteinases that are expressed in the insect gut. Most notable, SeCT34 contains a chymotrypsin activation site and the highly conserved motive DSGGP in the catalytic domain around the active-site serine is changed to DSGSA. Recombinant expression of SeCT34 was achieved in Sf21 insect cells using a special baculovirus vector, which has been engineered for optimized protein production. This is the first example of recombinant expression of an active serine proteinase which functions in the lepidopteran digestive tract. Purified recombinant SeCT34 enzyme was characterized by its ability to hydrolyze various synthetic substrates and its susceptibility to proteinase inhibitors. It appeared to be highly selective for substrates carrying a phenylalanine residue at the cleavage site. SeCT34 showed a pH-dependence and sensitivity to inhibitors, which is characteristic for semi-purified lepidopteran gut proteinases. Expression analysis revealed that SeCT34 was only expressed in the midgut of larvae at the end of their last instar, just before the onset of pupation. This suggests a possible role of this protein in the proteolytic remodelling that occurs in the gut during the larval to pupal molt.

Amino Acid Sequence↗
PubMed2005

Sequences of cDNAs and expression of genes encoding chitin synthase and chitinase in the midgut of Spodoptera frugiperda.

The focus of this study was on the characterization and expression of genes encoding enzymes responsible for the synthesis and degradation of chitin, chitin synthase (SfCHSB) and chitinase (SfCHI), respectively, in the midgut of the fall armyworm, Spodoptera frugiperda. Sequences of cDNAs for SfCHSB and SfCHI were determined by amplification of overlapping PCR fragments and the expression patterns of these two genes were analyzed during insect development by RT-PCR. SfCHSB encodes a protein of 1523 amino acids containing several transmembrane segments, whereas SfCHI encodes a protein of 555 amino acids composed of a catalytic domain, a linker region and a chitin-binding domain. SfCHSB is expressed in the midgut during the feeding stages, whereas SfCHI is expressed during the wandering and pupal stages. Both genes are expressed along the whole midgut. Chitin staining revealed that this polysaccharide is present in the peritrophic membrane (PM) only when SfCHSB is expressed. There is little or no chitin in the midgut when SfCHI is expressed. These results support the hypothesis that SfCHSB is responsible for PM chitin synthesis during the larval feeding stages and SfCHI carries out PM chitin degradation during larval-pupal molting, suggesting mutually exclusive temporal patterns of expression of these genes.

Amino Acid Sequence↗
PubMed2006

Biosynthesis of 10,12-dienoic fatty acids by a bifunctional Delta11 desaturase in Spodoptera littoralis.

In the biosynthetic pathway of Spodoptera littoralis sex pheromone, (E,E)-10,12-tetradecadienoic acid is produced from (Z)-11-tetradecenoic acid by desaturation and concomitant migration of the precursor double bond. With the aim of identifying the enzyme involved in this biotransformation, yeast Deltaelo1/Deltaole mutants, which are both elongase 1 and Delta9 desaturase-deficient, were transformed with the S. littoralis Delta11 desaturase gene using a Cu+2 inducible expression vector. The transformants produced a recombinant polyhistidine-tagged Delta11 desaturase that could be detected by immunoblotting from cell lysates. Lipid analysis revealed that besides producing large quantities of C11-monounsaturated fatty acids, mainly (Z)-11-hexadecenoic acid, (E,E)-10,12-tetradecadienoic acid and minor amounts of (E,Z)-10,12-hexadecadienoic acid were also produced, as well as very low quantities of another tetradecadienoate, which was tentatively identified as the (E,Z)-10,12-tetradecadienoic isomer. None of these dienes was detected with the Delta11 desaturase gene of Trichoplusia ni, which does not produce conjugated dienes as pheromone components. We conclude that the Delta11 desaturase of S. littoralis is a bifunctional enzyme with both Delta11 and Delta10,12 desaturation activities. The relationship between the substrate structure and the stereochemical outcome of the reaction is discussed.

Animals↗
PubMed2006

Expression and down-regulation of cytochrome P450 genes of the CYP4 family by ecdysteroid agonists in Spodoptera littoralis and Drosophila melanogaster.

The function of CYP4 genes in insects is poorly understood. Some CYP genes are up-regulated by ecdysteroids and a number of Cyp4 genes in Drosophila melanogaster have been shown by microarray to be down-regulated when the ecdysteroid titre is high, suggesting hormonal regulation. Here, we report the utilization of certain cloned CYP4 cDNAs/fragments to probe their developmental/tissue expression in the Lepidopteran, Spodoptera littoralis, including the effects of ecdysteroid receptor agonists (bis-acyl hydrazines). CYP4L8 is expressed essentially throughout the final larval instar of S. littoralis and, together with CYP4M12, is down-regulated by agonist. Furthermore, expression of these genes occurs in midgut, but is undetectable in brain, fat body, and integument. Similarly, in D. melanogaster, Cyp4ac1, Cyp4ac3, Cyp4ad1 and Cyp4d1 gene expression is drastically down-regulated by ecdysteroid agonist. The significance of the results is discussed in relation to the plausible functions of the CYP4 genes in Lepidoptera and mechanisms of down-regulation.

Animals↗
PubMed2003

Stable expression of recombinant human alpha3/4 fucosyltransferase III in Spodoptera frugiperda Sf9 cells.

Human alpha3/4 fucosyltransferase III (FT3; EC 2.4.1.65) synthesizes fucosylated glycoconjugates, namely the Lewis (Le) determinants. FT3 is detected in milk, gastric mucosa, kidney and other organs, but is found in very low amounts in these native tissues. In this work, we describe the expression of a soluble secretory form of FT3 (SFT3) in Spodoptera frugiperda (Sf9) insect cells using a non-lytic vector system. The coding sequence was cloned into the expression vector pIB/V5-His-TOPO which contains the transcriptional control of the Orgyia pseudotsugata multicapsid nucleopolyhedrosis virus immediate-early 2 (OpIE2) promoter. Transfected cells were selected using blasticidin-HCl. It was observed that the secreted activity SFT3 increased until the sixth day of culture when it reached the value 1.9 mU x 10(-6) cells and 13.4 mg/l, whereas only 5% of activity was retained inside the cells. Western blot analysis of secreted and intracellularly retained SFT3 had a similar variation. Comparison of the stable with the lytic baculovirus expression system showed that the former yielded approx. 13-fold more active SFT3, which was possibly due to a lower accumulation of intracellular SFT3.

Animals↗
PubMed2004

An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits the expression of an antibacterial peptide, cecropin, of the beet armyworm, Spodoptera exigua.

An entomopathogenic bacterium, Xenorhabdus nematophila, is known to depress hemocyte nodule formation of target insects by inhibiting eicosanoid biosynthesis. This study analyzed the inhibitory effect of X. nematophila on the humoral immunity of the target insects and tested its association with the host eicosanoid pathway. Plasma collected from the fifth instar larvae of Spodoptera exigua, when they were injected with X. nematophila, did not show antibacterial activity against Escherichia coli by a growth inhibition zone assay. In comparison, heat-killed X. nematophila induced significant antibacterial activity in the plasma. The antibacterial humoral activity was further demonstrated by examining a specific potent antibacterial peptide, cecropin. Two cecropin genes ('A' and 'B') were partially cloned from the fifth instar larvae of S. exigua by conserved degenerate primers using nested reverse transcriptase-polymerase chain reaction (RT-PCR). They showed high homologies with known cecropins from other lepidopteran species. Northern analysis using the cecropin probe showed that the injection of the heat-killed X. nematophila induced significant expression of a cecropin mRNA transcript (approximately 1.1 kb), but the larvae injected with the live bacteria did not show the corresponding transcript. Injection of arachidonic acid did not rescue the inhibition of X. nematophila based on either antibacterial activity or cecropin gene expression. The addition of dexamethasone, a specific phospholipase A2 inhibitor, did not inhibit antibacterial activity or cecropin gene expression when the larvae were injected with heat-killed X. nematophila. These results suggest that X. nematophila inhibits the antibacterial humoral immune reaction as well as the cellular immune reaction in S. exigua and that the inhibition of X. nematophila on the expression of the antibacterial peptide is not associated with inhibition of the eicosanoid pathway.

Analysis of Variance↗
PubMed2004

Effects of larval exposure to sublethal concentrations of the ecdysteroid agonists RH-5849 and tebufenozide (RH-5992) on male reproductive physiology in Spodoptera litura.

Sublethal concentrations of the bisacylhydrazine moulting hormone agonists, RH-5849, and tebufenozide (RH-5992) were fed to sixth (final) instar larvae of Spodoptera litura. Both RH-5849 and tebufenozide adversely affected the mating success of S. litura when the surviving treated males were crossed with normal females. The ecdysone agonists decreased the longevity of treated males and of untreated females when crossed with treated males. The number of eggs laid by untreated females mated to treated males was decreased, and the fertility (percentage of hatching success) of the resulting eggs was reduced. These effects on male reproductive success were at least in part explained by a reduction in the number of sperm transferred during mating. The adverse effects of tebufenozide on male reproductive function were qualitatively the same as those of RH-5849, but tebufenozide was active at lower concentrations. To understand the reason for these adverse effects on male reproduction, we investigated the effects of the insecticides on male reproductive physiology. Male reproductive tract development and testicular volume of resulting adult moths were adversely affected by sublethal larval exposure to the ecdysone agonists. Dose-dependent reductions occurred in the production of eupyrene and apyrene spermatozoa in the adult testes, and in the number of spermatozoa released from the testes into the male reproductive tract. The descent into the male tract of both eupyrene and apyrene sperm was found to start at the normal stage of development in both normal and treated insects, but the daily rhythm of sperm descent was subsequently disturbed in the insecticide-treated moths. This affected the numbers of sperm in the upper vas deferens (UVD), seminal vesicle (SV), and duplex (duplex). Injections of RH-5849 given to pharate adult or newly emerged adult S. litura also caused drastic reduction in the number of sperm in the upper regions of the male tract, when measured 24 h after injection. The possible importance of pest population reduction through the sublethal anti-reproductive effects of insecticides is discussed.

Animals↗
PubMed2004

Is the host or the parasitoid in control?: effects of host age and temperature on pseudoparasitization by Microplitis rufiventris in Spodoptera littoralis.

We compared the production of pseudoparasitization by Microplitis rufiventris females in most (third) and less (fourth) preferred instars of Spodoptera littoralis larvae at 20+/-1 and 27+/-1 degrees C. The parasitized hosts were classified into hosts producing parasitoids (type A hosts) and hosts producing no parasitoids, i.e., pseudoparasitized hosts (type B hosts). The latter were further classified into: (a) pseudoparasitized hosts with "well" arrested development (type B1 hosts); (b) pseudoparasitized hosts with partially arrested development (type B2 hosts); and (c) pseudoparasitized hosts that successfully pupated to apparently normal host pupae (type B3 hosts). The present series of experiments showed that parasitization by M. rufiventris was clearly affected by host instar, age within an instar and rearing temperature. Production of type B hosts was less when third instar S. littoralis larvae were exposed to the wasp females than when the host larvae were in fourth instar. The production of type A hosts was much greater when early or mid ages of an instar was stung by the wasp females comparing with stung late age of the same instar. Production of type B hosts may be due to one or overall of the following: (a) dosage dilution of M. rufiventris female's factors in the different age classes of the instar; (b) endocrine system (physiological state) at parasitization time, i.e., early vs late age of the instar; (c) growth rate of host larvae. The lowest production of type B hosts was at highest growth rate; and (d) temperature, larger proportions of type B hosts were produced at 27+/-1 than at 20+/-1degrees C. The three types host development (B1, B2 and B3) are possibly representing three levels of host resistance (host control) resulting in partial or complete failure of parasitoid control. Type A hosts represent complete success of parasitoid control. The results suggest that the impact of parasitoid factor(s) on developmental arrest is affected by host age at the time of parasitism and/or by temperature.

Age Factors↗
PubMed2004

A comparison of nutrient regulation between solitarious and gregarious phases of the specialist caterpillar, Spodoptera exempta (Walker).

Nutritional regulatory responses were compared between solitarious and gregarious phases of the African armyworm, Spodoptera exempta. When allowed to mix between two nutritionally imbalanced but complementary foods, final-instar caterpillars in both phases selected a diet comprising more carbohydrate than protein. This contrasts with other larval lepidopterans studied to date. Only minor differences were found in the position of the intake target for the two phases, despite their different energetic requirements for migration as adults. When restricted to nutritionally imbalanced diets, caterpillars of both phases were less disposed to overeat protein on high-protein diets than carbohydrate on high-carbohydrate diets, relative to the self-composed intake target. However, in both cases gregarious larvae overingested the excess nutrient to a greater degree than did solitarious larvae. Furthermore, gregarious larvae showed higher nitrogen conversion efficiency on an extreme protein-limiting diet, and accumulated more lipid per amount of carbohydrate consumed on carbohydrate-deficient diets. These phase-associated nutritional differences are consistent with the life-history strategies of the two phases.

Animals↗
PubMed2005

Modes of phagocytosis of Gram-positive and Gram-negative bacteria by Spodoptera littoralis granular haemocytes.

Haemocytes are the main immunocompetent cells in insect cellular immune reactions. Here, we show that in Spodoptera littoralis, granular haemocytes are the primary phagocyte haemocytes, both in vivo and in vitro. The "trigger" and "zipper" modes of engulfment known in mammal macrophages are active, in vivo, in S. littoralis granular haemocytes, together with macropinocytosis. Lipopolysaccharide as well as lipoteichoic acid inhibit the binding of both Gram-positive (Corynebacterium xerosis) and Gram-negative (Escherichia coli) bacteria on granular haemocytes. In addition, different ligands can inhibit the binding of E. coli. Most of these inhibitors are known as ligands of scavenger receptors in mammal macrophages and we hypothesise that one of the receptors present on S. littoralis granular haemocytes could be a scavenger-like receptor.

Animals↗
PubMed2005

Circadian mating activity and effect of pheromone pre-exposure on pheromone response rhythms in the moth Spodoptera littoralis.

Mating in moths is generally mediated by female-produced sex pheromones. Mating activity, female pheromone production/release and male pheromone responsiveness all show diurnal variations in many species. We found that the response of the male Egyptian cotton leafworm, Spodoptera littoralis, to sex pheromone gland extracts showed a diel rhythm in olfactometer tests, and the variation was persistent for at least 1 day in constant darkness. High male response to sex pheromone was correlated in time with high mating and locomotor activity. Male S. littoralis, maintained in constant darkness and exposed to pheromone gland extracts on a daily basis, showed an induced temporal variation in response after several days, in contrast to unexposed males. This suggests that in the absence of other external zeitgebers, exposure to sex pheromone may function to synchronise circadian behavioural rhythms in male moths. The daily rhythm in mating activity in S. littoralis is also shown to be persistent for at least 2 days in constant darkness. Pairs mated significantly less when either the male or female had been raised in a light:dark cycle 10 h out of phase, indicating that the proposed circadian rhythm in mating activity is composed of rhythmic mating preference/ability in both sexes.

Animals↗
PubMed2005

The calyx fluid of Microplitis rufiventris parasitoid and growth of its host Spodoptera littoralis larvae.

The morphology of the female reproductive system of pupal and adult stages of Microplitis rufiventris and the ultrastructure of the ovaries are described and illustrated. Two morphologically distinct types of particles of nuclear origin, i.e., polydnavirus (PDV) and a virus-like filamentous particle (VLFP) were detected in the ovarian calyx fluid of the female wasp. It is likely that these particles are injected into the host during oviposition. PDV initiated replication in the calyx of mid-aged pupae and in pharate adults and were present throughout adult life. VLFP were only seen in the calyx fluid of newly emerged adults, and therefore observed after the PDV. Feulgen and methyl-green pyronin staining revealed the presence of DNA in both types of particles. The effects of injection of Spodoptera littoralis larvae with a combination of parasitoid viruses and venom of M. rufiventris females (CxFV) were investigated and the results were compared with two control groups, i.e., larvae injected with Pringle's saline (PS) and naturally parasitized larvae. CxFV-larvae showed significant declines (P<0.05) in food consumption, weight of ejected faeces and weight gain when compared with PS-larvae. However, naturally parasitized larvae (parasitoid egg+CxFV+ovarian protein) displayed a high significance score (P<0.01) in comparison with those of PS-larvae. The approximate digestibility (AD) values of S. littoralis larvae were positively affected as early as day 2 post-treatment by either injection of CxFV or parasitization. However, a reduction in AD was observed in both PS- and CxFV-larvae on day 3-7 in comparison with naturally parasitized larvae. Other indices of food utilization were unchanged in CxFV-larvae when compared to saline treated or parasitized controls.

Animals↗