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Changes in the haemolymph proteome of Spodoptera littoralis induced by the parasitoid Chelonus inanitus or its polydnavirus and physiological implications.

The egg-larval parasitoid Chelonus inanitus induces in its host Spodoptera littoralis two major developmental effects, namely a precocious onset of metamorphosis followed by a developmental arrest in the prepupal stage. Along with each egg, the wasp injects polydnavirus and venom into the host egg. The polydnavirus has been shown to play a major role in inducing the developmental arrest while the parasitoid larva is instrumental in inducing the precocious onset of metamorphosis. Here we report that experimental dilution of haemolymph of polydnavirus-containing larvae can partially prevent the developmental arrest while injection of native, but not of heat-treated, haemolymph or plasma from polydnavirus-containing larvae into nonparasitized larvae could induce developmental arrest in 14-15% of the larvae. This illustrates that heat-labile factors present in haemolymph play a role in causing developmental arrest. Injection of parasitoid medium increased the proportion of larvae entering metamorphosis precociously while injection of antibodies against a parasitoid-released protein had the opposite effect; this indicates that this protein and possibly other parasitoid-released substances are involved in inducing the precocious onset of metamorphosis. Analysis of the plasma proteome of nonparasitized, parasitized and polydnavirus-containing larvae revealed that the developmental effects are associated with only minor differences: eleven low abundant viral or virus-induced proteins and five parasitoid-released proteins were seen at specific stages of the host.

Animals↗

Distribution and residual activity of two insecticidal proteins, avidin and aprotinin, expressed in transgenic tobacco plants, in the bodies and frass of Spodoptera litura larvae following feeding.

To understand how a major cosmopolitan pest responds to two very different insecticidal proteins and to determine whether herbivorous insects and their frass could be environmental sources of recombinant proteins from transgenic plants, Spodoptera litura (Fab.) (Lepidoptera, Noctuidae) larvae were fed on tobacco leaves expressing either the biotin-binding protein, avidin, or the protease inhibitor, aprotinin. Control larvae received non-transgenic tobacco. Samples of larvae were taken after 5, 6 or 7 days' feeding and frass was collected after two 24-h periods at 6 and 7 days. Insects in all treatments grew significantly during the experiment, but the avidin-fed larvae were significantly smaller than the others on Day 7. Avidin was found in all samples of avidin-fed larvae (7.0+/-0.86 ng mg(-1), n=45), at a lower level than in their frass (31.9+/-5.08 ng mg(-1), n=30), and these frass levels were lower than those of the the leaves fed to the larvae (69.0+/-6.71 ng mg(-1), n=45). All of the avidin detected in these samples was capable of binding biotin. On average, between 10 and 28% of avidin was recovered with the methods used, whereas almost full recovery of aprotinin was effected. Aprotinin levels in larvae (8.2+/-0.53 ng mg(-1), n=45) were also lower than aprotinin levels in frass (77.4+/-6.9 ng mg(-1), n=30), which were somewhat lower than those in the leaves fed to the larvae (88.6+/-2.51 ng mg(-1), n=45). Approximately half the trypsin-binding ability of aprotinin was lost in larvae, and in frass, aprotinin had lost about 90% of its ability to bind trypsin.

Animals↗

Antioxidant enzymes in Spodoptera littoralis (Boisduval): are they enhanced to protect gut tissues during oxidative stress?

The Egyptian armyworm Spodoptera littoralis is a polyphagous insect attacking a number of plant species including those belonging to the Solanaceae and Cruciferaceae families. Its digestive physiology must therefore adapt to the food plant to ensure maximum extraction of nutrients with minimum trade-off in terms of growth retardation by pro-oxidant allelochemicals. To investigate this, the caterpillars of S. littoralis were fed on a semi-artificial diet (Manduca Premix-Heliothis Premix) and for 24 h on potato plants (Solanum tuberosum), respectively, at the mature 6th instar, and the levels of oxidative radicals and antioxidant enzymes in their guts were compared. The gut pH, standard redox potential (Eh) and electron availability (pe) revealed that oxidizing conditions prevail which promote oxidation of pro-oxidant allelochemicals in foliage. Oxidative stress in the foregut and midgut tissue and the gut contents was assessed from the generation of superoxide radical, total peroxide content and protein carbonyl content. Antioxidant defense was measured by the activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APOX) and glutathione S-transferase peroxidase (GSTpx). A significant (p < 0.001) increase in the superoxide radical production (in foregut tissue, foregut and midgut contents), concomitant with an increase in total peroxide (in foregut contents) and protein carbonyl levels (in foregut and midgut tissue) were noted in larvae fed on the plants in contrast to those fed the semi-artificial diet. Similarly, a significant up-regulation of antioxidant enzymes SOD (in midgut tissues), CAT (in foregut, midgut tissue and contents), APOX (in foregut contents, midgut tissue and contents) and GSTpx (in foregut tissues) was recorded on the plant diet in comparison to the semi-artificial diet. The pro-oxidant allelochemicals in the plant diet are thus eliminated by the insect at the expense of up-regulation of antioxidative enzymes in response to increased oxidative stress from oxidizable allelochemicals. The results are consistent with the hypothesis that increased concentrations of antioxidants form an important component of the defense of herbivorous insects against both exogenous and endogenous oxidative radicals.

Animals↗

Cytotoxicity of fungal metabolites to lepidopteran (Spodoptera frugiperda) cell line (SF-9).

The toxicity of sixteen fungal metabolites produced by some entomopathogenic fungi or biological control fungi agents was evaluated on lepidopteran Spodoptera frugiperda (SF-9) cell line by Trypan blue dye exclusion and MTT-colorimetric assay, after 48 h of incubation. No statistical difference was found between IC50values (50% Inhibiting Concentration) and CC50 values (50% Cytotoxicity Concentration) obtained by MTT test and Trypan blue dye exclusion for each fungal metabolite. By MTT assay, the cytotoxicity ranking was fusarenon X (IC50 0.3 microM) = diacetoxyscirpenol (IC50 0.5 microM) = beauvericin (IC50 2.5 microM) = nivalenol (IC50 5.3 microM) = enniatin (IC50 6.6 microM) > or = gliotoxin (IC50 7.5 microM) > zearalenone (IC50 17.5 microM) > deoxynivalenol (IC50 47.6 microM). By Trypan blue dye exclusion the cytotoxicity ranking was fusarenon X (CC50 0.4 microM) = diacetoxyscirpenol (CC50 1.1 microM) beauvericin = (CC50 3.0 microM)=gliotoxin (CC50 4.0 microM) = enniatin (CC50 6.7 microM) > or = nivalenol (CC50 9.5 microM) > zearalenone (CC50 18.3 microM) > deoxynivalenol (CC50 45.0 microM). The comparison with other bioassays showed that the SF-9 insect cell line could represent a further tool to screen for the toxic effects of fungal metabolites especially for beauvericin, gliotoxin, and zearalenone.

Animals↗

Occurrence and characterization of a nucleopolyhedrovirus from Spodoptera littoralis (Lepidoptera: Noctuidae) isolated in the azores.

A nucleopolyhedrovirus (SpliMNPV-Az) was isolated from diseased larvae of Spodoptera littoralis, collected at the Island of S. Miguel in Azores. The virulence of this isolate was tested against S. littoralis larvae in laboratory. LD50 against 2nd and 3rd instars were not significantly different, 1.44x10(4), 3.89x10(4) OBs per larvae, respectively, but both were significantly different from that against 4th instar, which was 61.3x10(4) OBs per larvae. The complete codons sequence of SpliMNPV-Az Polh gene obtained was 750 bp (NCBI GenBank Accession No. AY600451). This sequence was compared to other 38 polyhedrin genes from NPVs and to 6 granulin genes from GVs and resulted to be identical to the sequence of a SpliMNPV previously published, thus indicating that the natural host of SpliMNPV-Az must be S. littoralis. Genetic distances estimated from restriction enzymes profiles showed SpliMNPV-Az is close to the Egyptian SpliMNPV type B, despite some degree of genetic divergence suggested by slight differences observed on PstI profile.

Amino Acid Sequence↗

New cell lines from larval fat bodies of Spodoptera exigua: characterization and susceptibility to baculoviruses (Lepidoptera: Noctuidae).

Two new cell lines, designated IOZCAS-Spex-II and IOZCAS-Spex-III, were initiated from the fat bodies of larvae of the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae) in TNM-FH medium containing 10% fetal bovine serum. The spherical cells were predominant among the various cell types and measures approximately 15 microm in diameter. The cell lines were mainly composed of tetraploid cells with chromosome numbers ranging from 116 to 131 (n=31). The cell lines were confirmed to have originated from the S. exigua by DAF-PCR technique. They were susceptible to the multiple nucleocapsid nuclear polyhedrosis viruses from S. exigua.

Animals↗

Direct infection of Spodoptera litura by Photorhabdus luminescens encapsulated in alginate beads.

Actively growing cultures of Photorhabdus luminescens were encapsulated in sodium alginate beads and examined for their ability to infect insect hosts. These beads, containing approximately 2.5 x 10(7)Photorhabdus cells per bead, when mixed with sterilized soil and exposed to Spodoptera litura larvae resulted in 100% mortality in 48 h, while the use of alginate encapsulated Heterorhabditis nematode resulted in 40% mortality after 72 h. The bacteria were reisolated from the dead insect thus proving Koch's postulates and demonstrating the ability of P. luminescens to kill the insect host on their own, independent of the symbiont nematode. The LC(50) dose of Photorhabdus cells was estimated at 1010 cells per larva for killing S. litura 6th instar larvae in 48 h.

Alginates↗

Application of the PCR-RFLP method for the rapid differentiation of Spodoptera exigua nucleopolyhedrovirus genotypes.

Quality control during mass production of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV), and studies on environmental fate following the use of this virus as a biological pesticide, would be facilitated by a rapid method for the detection and identification of isolates. A molecular biology tool was developed that combined the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) to differentiate SeMNPV isolates. Oligonucleotide primers were designed to amplify five variable SeMNPV genomic regions (V01, V02, V03, V04, V05). Four wild-type SeMNPV strains isolated from the United States (US2) and Spain (SP1, SP2, and SP3), and a laboratory cloned genotype (US1A), were analyzed with 36 different primer-endonuclease combinations. BglII digestion of the variable region 1 (V01) amplicon was the only combination that differentiated each of the five virus isolates tested, although genetic heterogeneity limited the discriminatory power of the technique. Six novel SeMNPV isolates originating from greenhouse soils in southern Spain were successfully identified using this method. As judged by sequence analysis, the V01 region, which comprises the homologous region 1 (hr1), is the most variable genomic region among the genotypes present in the Spanish isolates. This method constitutes a useful tool for processing large number of environmental samples and could be used to address environmental biosafety concerns.

Animals↗

Preliminary analysis of the GABA-induced current in cultured CNS neurons of the cutworm moth, Spodoptera litura.

Properties of GABA-induced current in cultured CNS (ganglion) neurons of cutworm moths (Spodoptera litura) were studied using a whole-cell patch-clamp technique. CNS neurons ranging from 10 to 20 microm in diameter were cultured for 4-7 days in MGM-464 medium. GABA-induced a current response in these neurons in a sigmoidally dose dependent manner where the Hill coefficient and EC50 were 2.2 and 33.0 microM, respectively. The reversal potential of GABA-induced current was -2.5 mV, which is close to the Cl- equilibrium potential that was calculated from chloride ion concentrations in the present experimental environment. Furthermore, the GABA-induced current response depended on the extracellular chloride ion concentration, indicating that the receptor regulates the Cl- permeability of cells. The GABA-induced current was completely inhibited by the GABA(A) antagonist, SR95531, and activated by the GABA(A) agonist, muscimol but not by the GABA(B) agonist, baclofen. On the other hand, the GABA(C) agonist, CACA, also induced a little smaller current than the GABA-induced response. The pharmacological behaviors of the GABA-induced currents suggest that these cells contain GABA receptors that belong to the GABA receptor family including the Rdl GABA receptors identified in Drosophila neurons.

Animals↗

Production and purification of functional truncated soluble forms of human recombinant L1 cell adhesion glycoprotein from Spodoptera frugiperda Sf9 cells.

L1 is a human cell adhesion glycoprotein involved in the development of the central nervous system that comprises six immunoglobulin-like domains (Ig1-Ig6), five fibronectin-type III (FN1-FN5) domains, a single transmembrane region and a cytoplasmic domain. It contains 20 potential N-glycosylation sites and is heavily glycosylated in a variety of cell types. In this work, seven truncated soluble forms including L1 ectodomain (L1/ECD) and Ig domains 5-6 (L1/Ig5-6) have been constructed by PCR and have been cloned, as well as the full-length form (L1), in the stable expression vector for insect cells pMIB/V5-His-TOPO. Spodoptera frugiperda Sf9 cell lines expressing the truncated forms have been obtained, and all proteins were successfully secreted. L1/ECD and L1/Ig5-6 were produced in shake flasks with productions of 3 and 32 mg/L on the third and fourth day of culture, respectively. When L1/Ig5-6 was produced for four days in 2L bioreactor 200 mg/L protein were recovered from the supernatants on the fourth day of culture. Affinity-purified L1/ECD and L1/Ig5-6 were immobilized on poly-d-lysine coated coverslips, and were shown to be active in inducing neurite outgrowth from human NT2N neurons. Therefore, correctly folded and functional truncated forms of human L1 have been produced in high amounts from insect cells using a stable expression system.

Animals↗

Presence of angiotensin converting enzyme isoforms in larval lepidoptera (Spodoptera littoralis).

In this research the presence of angiotensin converting enzyme (ACE) in larvae of the lepidopteran Spodoptera littoralis was evaluated. Making use of the substrate Abz-FRK-(Dnp)P-OH and the specific inhibitor captopril at 10 microM, ACE activity was determined in a fluorescence assay for intact larvae, hemolymph, head, midgut and dorsal tissue. In dorsal tissue and hemolymph, ACE activity was highest. These data are consistent with a possible role for ACE in contractions of the dorsal vessel and metabolism of circulating peptide hormones in the hemolymph. After the presence of ACE was confirmed, a sequential procedure of anion exchange and size exclusion chromatography was applied to purify ACE from whole wandering larvae (last stage). With this procedure, three different ACE pools were collected that cleaved the fluorogenic substrate Abz-FRK-(Dnp)P-OH. Activity could be inhibited by a final concentration of 2.5 microM captopril. In addition, two out of three samples eluted at different salt concentration and thus ACE 1, 2 and 3 represent at least two different ACE isoforms. These data reveal that ACE is present in S. littoralis and that at least two out of three isolated ACE forms are truly isoforms.

Angiotensin-Converting Enzyme Inhibitors↗

Insect growth regulatory effects of some extracts and sterols from Myrtillocactus geometrizans (Cactaceae) against Spodoptera frugiperda and Tenebrio molitor.

A methanol extract from the roots and aerial parts of Myrtillocactus geometrizans (Cactaceae) yielded peniocerol 1, macdougallin 2, and chichipegenin 3. The natural products 1, 2 their mixtures, MeOH and CH(2)Cl(2) extracts showed insecticidal and insect growth regulatory activity against fall armyworm [Spodoptera frugiperda J. E. Smith (Lepidoptera: Noctuidae)], an important insect pest of corn, and [Tenebrio molitor (Coleoptera)], a pest of stored grains in Mexico. The most active compounds were 1, 2, and a mixture (M(2)) of 1 and 2 (6:4). All these extracts, compounds and the mixture had insect growth regulating (IGR) activity between 5.0 and 50.0 ppm and insecticidal effects between 50 and 300 ppm in diets. The extracts were insecticidal to larvae, with lethal doses between 100 and 200 ppm. These compounds appear to have selective effects on the pre-emergence metabolism of Coleoptera, because in all treatments of the larvae of T. molitor, pupation were shortened and this process show precociousness in relation to controls. In contrast to S. frugiperda larvae, onset of pupation was noticeably delayed. Emergence in both cases was drastically diminished. In both pupae and in the few adults that were able to emerge, many deformations were observed. The results of these assays indicated that the compounds were more active than other known natural insect growth inhibitors such as gedunin and methanol extracts of Cedrela salvadorensis and Yucca periculosa. Peniocerol, macdougallin and chichipegenin, as well as mixtures of these substances, may be useful as natural insecticidal agents.

Animals↗

Characterization of a novel peptide with allatotropic activity in the fall armyworm Spodoptera frugiperda.

A cDNA that encodes 53 amino acids, including one copy of the RVRGNPISCF-OH peptide, was cloned from Spodoptera frugiperda. This peptide strongly stimulates the synthesis and release of juvenile hormone (JH) in vitro by the corpora allata (CA) of S. frugiperda and was code-named Spofr-AT 2. Northern blotting and reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed that the preprohormone is expressed as one transcript in the brain, midgut (Mg) and ovary (Ov) in a tissue- and developmental-specific manner. Whole-mount in situ hybridization confirmed the gene expression in the suboesophageal ganglion (SOG) and in the ovary of adult females. Treating the CA with the synthetic peptide caused an up to tenfold increase in the release of JH. The stimulation was dose-dependent with an apparent EC(50) of ca. 10(-7) M. CA that were activated with Spofr-AT 2 could be inhibited by the addition of synthetic allatostatin type-C from Manduca sexta (Manse-AS). This is the first report on the presence and function of two different peptides with allatotropic activity in an insect species.

Amino Acid Sequence↗

Function of Spodoptera exigua nucleopolyhedrovirus late gene expression factors in the insect cell line SF-21.

We used a well established transient expression assay to test the ability of the baculovirus Spodoptera exigua M nucleopolyhedrovirus (SeMNPV) homologs of Autographa californica MNPV (AcMNPV) late expression factors (lefs) to activate a late promoter-reporter gene cassette in SF-21 cells. This insect-derived cell line is fully permissive for AcMNPV infection but not for SeMNPV. In the assay, 19 AcMNPV lefs stimulate optimal levels of late gene promoter activity. SeMNPV lef-5 successfully replaced the corresponding AcMNPV gene in the context of the remaining set of AcMNPV lefs, whereas SeMNPV dnapol and 39k exhibited partial activity. When all the SeMNPV lefs were assayed together or in the presence of four lefs encoded only in AcMNPV, it resulted in background levels of late promoter-driven reporter gene activity. However, SeMNPV genomic DNA and the four AcMNPV-specific lefs stimulated low levels of reporter gene activity. Moreover, SeMNPV IE-1, but not AcMNPV IE-1, further stimulated late gene expression in the presence of SeMNPV DNA. AcMNPV IE-1 was able to mediate early gene expression cis-linked to homologous regions (hrs) derived from AcMNPV and SeMNPV. In contrast, SeMNPV IE-1 was more specific for SeMNPV-derived hr elements.

Animals↗

Characterization of gp41 gene of Spodoptera litura multicapsid nucleopolyhedrovirus.

Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) gp41 gene is 993 bp long and the protein encoded by this gene has 6-66% amino acid identities with other known baculovirus GP41 proteins. Slgp41 transcripts were detected from 12 to 96 h post-infection (p.i.) and the mRNA start site was mapped within a consensus baculovirus late promoter sequence (ATAAG). Western blot analysis of extracts from SpltMNPV-infected S. litura cells detected a 41 kDa protein, and this protein was present in the nucleus of infected cells from 12 to 96 h p.i., whereas in the cytoplasm from 24 to 96 h p.i. Structural localization confirmed that SlGP41 is associated with the envelope of occlusion-derived virus (ODV). Lectin-binding assay showed that three lectins erythrina cristaglli lectin (ECL), lycopersicon esculentum lectin (LEL), and bandeiraea simlicifolia lectin (BSL) recognizing N-acetylglucosamine were specifically bound to SlGP41. It was proposed that SlGP41 is an O-glycoprotein.

Amino Acid Sequence↗

Characterization of Spodoptera litura multicapsid nucleopolyhedrovirus 38.7 k protein, which contains a conserved BRO domain.

Homology analysis revealed that Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) 38.7 k protein has 22-83% amino acid identities with Ecotropis obliqua NPV, Mamestra configurata MNPV, Helicoverpa armigera SNPV, H. zea SNPV, S. exigua MNPV and S. littoralis MNPV 38.7 k proteins. Analysis of the relationship of these 38.7 k proteins indicated that they contain a conserved BRO-N domain, and SpltMNPV and SpliMNPV 38.7 k proteins also contain a motif found in all known viral and prokaryotic single-strand DNA binding proteins. RT-PCR results showed that SpltMNPV 38.7 k gene is transcribed actively at the late stage of infection and the mRNA start site was mapped within a consensus baculovirus late promoter motif (ATAAG). Western blot analysis revealed that the 38.7 k was expressed in infected S. litura cells as a 41 kDa form and this protein distributed in the nucleus of infected cells. Using a histone extraction protocol, SpltMNPV 38.7 k could be detected in the histone H1 fraction. Micrococcal nuclease treatment released SpltMNPV 38.7 k protein from the chromatin fraction, suggesting that its involvement in nucleosome structures. Furthermore, column chromatography using DNA-cellulose showed that SpltMNPV 38.7 k protein interacted with nucleic acids. It was proposed that SpltMNPV 38.7 k might function as a DNA-binding protein.

Amino Acid Motifs↗

Characterization of Spodoptera exigua multicapsid nucleopolyhedrovirus ORF100 and ORF101, two homologues of E. coli ChaB.

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. The baculovirus Spodoptera exigua multicapsid nucleopolyhedrovirus contains two homologues of E. coli ChaB, open reading frames (ORFs) 100 (Se100) and 101 (Se101). RT-PCR and 5' RACE analyses indicated that transcription of both SeMNPV chaB genes occurs by 24 h postinfection and is initiated at a late consensus ATAAG motif. Immunoblot analysis showed that Se100 and Se101 were, respectively, expressed as 11 and 28kDa proteins in infected cells, and they were distributed in both cytoplasm and nucleus. Further analysis revealed that Se100 and Se101 are associated with nuclear structure and only present in the nucleocapsid of occlusion-derived virus. Additionally, column chromatography analysis showed that both proteins could weakly interact with nucleic acids. It was proposed that SeMNPV ChaB might function as DNA binding proteins.

Amino Acid Sequence↗

Lipid composition of Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn) insect cells used for baculovirus infection.

The lipid composition of two different insect cell lines from Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn) which are established cell lines for infection with recombinant baculovirus was analyzed by high-performance liquid chromatography and gas-liquid chromatography. The major phospholipids found were phosphatidylcholine and phosphatidylethanolamine, the major mono-unsaturated fatty acids were oleic acid and palmitoleic acid, the major saturated fatty acid was stearic acid. The cholesterol to phospholipid ratio was demonstrated to be lower than in mammalian cell lines. Infection with a recombinant baculovirus Autographa californica resulted in increased levels of phosphatidylcholine in the insect cells. The baculovirus/insect cell system has become a popular system for heterologous protein production. Functional changes of membrane proteins produced in these two cell lines might be correlated to a different lipid profile of their cellular membranes.

Animals↗