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PubMed2005

Bacillus thuringiensis Cry1Ca-resistant Spodoptera exigua lacks expression of one of four Aminopeptidase N genes.

BACKGROUND: Insecticidal toxins from Bacillus thuringiensis bind to receptors on midgut epithelial cells of susceptible insect larvae. Aminopeptidases N (APNs) from several insect species have been shown to be putative receptors for these toxins. Here we report the cloning and expression analysis of four APN cDNAs from Spodoptera exigua. RESULTS: Suppression Subtractive Hybridization (SSH) was used to construct cDNA libraries of genes that are up-and down-regulated in the midgut of last instar larvae of beet armyworm, S. exigua exposed to B. thuringiensis Cry1Ca toxin. Among the clones from the SSH libraries, cDNA fragments coding for two different APNs were obtained (APN2 and APN4). A similar procedure was employed to compare mRNA differences between susceptible and Cry1Ca resistant S. exigua. Among the clones from this last comparison, cDNA fragments belonging to a third APN (APN1) were detected. Using sequences obtained from the three APN cDNA fragments and degenerate primers for a fourth APN (APN3), the full length sequences of four S. exigua APN cDNAs were obtained. Northern blot analysis of expression of the four APNs showed complete absence of APN1 expression in the resistant insects, while the other three APNs showed similar expression levels in the resistant and susceptible insects. CONCLUSION: We have cloned and characterized four different midgut APN cDNAs from S. exigua. Expression analysis revealed the lack of expression of one of these APNs in the larvae of a Cry1Ca-resistant colony. Combined with previous evidence that shows the importance of APN in the mode of action of B. thuringiensis toxins, these results suggest that the lack of APN1 expression plays a role in the resistance to Cry1Ca in this S. exigua colony.

Amino Acid Sequence↗
PubMed2006

Gene expression profiling of Spodoptera frugiperda hemocytes and fat body using cDNA microarray reveals polydnavirus-associated variations in lepidopteran host genes transcript levels.

BACKGROUND: Genomic approaches provide unique opportunities to study interactions of insects with their pathogens. We developed a cDNA microarray to analyze the gene transcription profile of the lepidopteran pest Spodoptera frugiperda in response to injection of the polydnavirus HdIV associated with the ichneumonid wasp Hyposoter didymator. Polydnaviruses are associated with parasitic ichneumonoid wasps and are required for their development within the lepidopteran host, in which they act as potent immunosuppressive pathogens. In this study, we analyzed transcriptional variations in the two main effectors of the insect immune response, the hemocytes and the fat body, after injection of filter-purified HdIV. RESULTS: Results show that 24 hours post-injection, about 4% of the 1750 arrayed host genes display changes in their transcript levels with a large proportion (76%) showing a decrease. As a comparison, in S. frugiperda fat body, after injection of the pathogenic JcDNV densovirus, 8 genes display significant changes in their transcript level. They differ from the 7 affected by HdIV and, as opposed to HdIV injection, are all up-regulated. Interestingly, several of the genes that are modulated by HdIV injection have been shown to be involved in lepidopteran innate immunity. Levels of transcripts related to calreticulin, prophenoloxidase-activating enzyme, immulectin-2 and a novel lepidopteran scavenger receptor are decreased in hemocytes of HdIV-injected caterpillars. This was confirmed by quantitative RT-PCR analysis but not observed after injection of heat-inactivated HdIV. Conversely, an increased level of transcripts was found for a galactose-binding lectin and, surprisingly, for the prophenoloxidase subunits. The results obtained suggest that HdIV injection affects transcript levels of genes encoding different components of the host immune response (non-self recognition, humoral and cellular responses). CONCLUSION: This analysis of the host-polydnavirus interactions by a microarray approach indicates that the presence of HdIV induces, directly or indirectly, variations in transcript levels of specific host genes, changes that could be responsible in part for the alterations observed in the parasitized host physiology. Development of such global approaches will allow a better understanding of the strategies employed by parasites to manipulate their host physiology, and will permit the identification of potential targets of the immunosuppressive polydnaviruses.

Animals↗
PubMed2006

Analysis and functional annotation of expressed sequence tags from the fall armyworm Spodoptera frugiperda.

BACKGROUND: Little is known about the genome sequences of lepidopteran insects, although this group of insects has been studied extensively in the fields of endocrinology, development, immunity, and pathogen-host interactions. In addition, cell lines derived from Spodoptera frugiperda and other lepidopteran insects are routinely used for baculovirus foreign gene expression. This study reports the results of an expressed sequence tag (EST) sequencing project in cells from the lepidopteran insect S. frugiperda, the fall armyworm. RESULTS: We have constructed an EST database using two cDNA libraries from the S. frugiperda-derived cell line, SF-21. The database consists of 2,367 ESTs which were assembled into 244 contigs and 951 singlets for a total of 1,195 unique sequences. CONCLUSION: S. frugiperda is an agriculturally important pest insect and genomic information will be instrumental for establishing initial transcriptional profiling and gene function studies, and for obtaining information about genes manipulated during infections by insect pathogens such as baculoviruses.

Animals↗
PubMed2006

Members of the Hyposoter didymator Ichnovirus repeat element gene family are differentially expressed in Spodoptera frugiperda.

BACKGROUND: The abundance and the conservation of the repeated element (rep) genes in Ichnoviruses genomes suggest that this gene family plays an important role in viral cycles. In the Ichnovirus associated with the wasp Hyposoter didymator, named HdIV, 10 rep genes were identified to date. In this work, we report a relative quantitative transcription study of these HdIV rep genes in several tissues of the lepidopteran host Spodoptera frugiperda as well as in the H. didymator wasps. RESULTS: The data obtained in this work indicate that, in the early phases of infection (24 hours), HdIV rep genes each display different levels of transcripts in parasitized 2nd instar or HdIV-injected last instar S. frugiperda larvae. Only one, rep1, is significantly transcribed in female wasps. Transcript levels of the HdIV rep genes were found as not correlated to their copy number in HdIV genome. Our results also show that HdIV rep genes display different tissue specificity, and that they are primarily transcribed in S. frugiperda fat body and cuticular epithelium. CONCLUSION: This work is the first quantitative analysis of transcription of the ichnovirus rep gene family, and the first investigation on a correlation between transcript levels and gene copy numbers in Ichnoviruses. Our data indicate that, despite similar gene copy numbers, not all the members of this gene family are significantly transcribed 24 hours after infection in lepidopteran larvae. Additionally, our data show that, as opposed to other described HdIV genes, rep genes are little transcribed in hemocytes, thus suggesting that they are not directly associated with the disruption of the immune response but rather involved in other physiological alterations of the infected lepidopteran larva.

Amino Acid Sequence↗
PubMed2005

Cloning and characterization of ecdysone receptor and ultraspiracle cDNAs from Spodoptera litura.

The insect molting hormone, ecdysone, induces a series of events critical for ecdysis, beginning with the formation of a complex between ecdysteroid receptor (EcR) and its partner protein, ultraspiracle (Usp). In the present study, we cloned cDNAs encoding EcR and Usp by RT-PCR and 5'-/3'-RACE from the common cutworm, Spodoptera litura, a destructive agricultural pest insect in East Asia. Hexahistidine-tagged recombinant EcR and Usp of S. litura were expressed in E. coli, and the proteins, after refolding, exhibited the specific binding to an ecdysone analogue in vitro.

Animals↗
PubMed2002

Glomerular representation of plant volatiles and sex pheromone components in the antennal lobe of the female Spodoptera littoralis.

We studied the projection patterns of antennal lobe (AL) interneurones sensitive to plant volatiles and female-produced sex pheromone components in the female moth, Spodoptera littoralis. Ten compounds (eight plant-derived compounds and two sex pheromone components) were singly applied to the antenna and, using intracellular recording and staining techniques, the physiological and morphological characteristics of responding neurones were investigated. In addition, ALs stained with a synapsin antibody were optically sectioned using confocal microscopy, and a three-dimensional map of glomeruli in the anterior aspect of the AL was reconstructed. We used the map as a reference for identification of glomeruli innervated by projection neurones (PNs) that respond to plant volatiles and/or pheromone components. Nineteen PNs, responding to one to seven compounds of the ten tested stimuli, were stained with neurobiotin. These neurones each arborised in a single glomerulus in the frontal side of the AL. PNs responding to the same compound arborised in different glomeruli and PNs arborising in the same glomerulus responded to different compounds. Accordingly, glomeruli harbouring the dendritic arborisations of PNs responding to each of the tested compounds constituted a unique array of glomeruli that were not necessarily adjacent. It was thus clear that, at the output level, a single plant volatile or a sex pheromone component was not represented within a single glomerulus in the AL. We expect complex patterns of glomeruli to be involved in the coding of plant-derived compounds, as well as sex pheromone components, in female S. littoralis.

Animals↗
PubMed2004

Biotransformation of sinesetin by the larvae of the common cutworm (Spodoptera litura).

Biotransformation of sinesetin (1) by larvae of Spodoptera litura was investigated. Compound 1 was converted to a new flavonoid, (+)-5,7,3',4'-tetramethoxyflavone-6-O-beta-D-glucoside (4), and two known flavones, 4'-hydroxy-5,6,7,3'-tetramethoxyflavone (2) and 6-hydroxy-5,7,3',4'-tetramethoxyflavone (3). These structures were established by IR, HR-EI-MS, HR-FAB-MS, 1D NMR, and 2D NMR spectral studies. The results indicate that the metabolic reaction of compound 1 by larvae of S. litura proceeded along two pathways; the main pathway is demethylation at the C-6 position followed by glucosylation, and the minor pathway is demethylation at the C-4' position.

Animals↗
PubMed2006

Biotransformation of isoflavones by the larvae of the common cutworm (Spodoptera litura).

Biotransformation of the 5,7,4'-trimethoxyisoflavone (1), 6,7,4'-trimethoxyisoflavone (2), and 7,4'-dimethoxyisoflavone (3) by insects, Spodoptera litura was investigated. Compound 1 was transformed to 5-hydroxy-7,4'-dimethoxyisoflavone (4), 7-hydroxy-5,4'-dimethoxyisoflavone (5) and 4'-hydroxy-5,7-dimethoxyisoflavone (6) by S. litura. Compounds 2 and 3 were hardly metabolized by S. litura. This suggested that compound 1 was converted to compounds 4, 5 and 6 by demethylation at the C-5, C-7 and C-4' position, respectively.

Animals↗
PubMed1999

Green-tissue-specific expression of a reconstructed cry1C gene encoding the active fragment of Bacillus thuringiensis delta-endotoxin in haploid tobacco plants conferring resistance to Spodoptera litura.

The DNA sequence of a truncated cry1C gene encoding the active fragment of Bacillus thuringiensis (Bt) delta-endotoxin was fully reconstructed by introduction of silent mutations. Each of the truncated wild type and the synthetic genes encoding the active fragment of the protoxin was introduced into haploid tobacco plants under the control of the rbcS promoter. To facilitate selection of transgenic tobacco plants with high insecticidal activity, a fusion gene encoding both rat CYP1A1 cytochrome P450 and yeast NADPH-P450 oxidoreductase was cotransformed with the wild type cry1C gene. The synthetic gene elevated the levels of Cry1C protein and the mRNA in transgenic tobacco plants as well as mortality in Spodoptera litura larvae. The Cry1C protein was accumulated mainly in the leaf tissues of the transgenic tobacco plants. The results reported here imply that the green-tissue-specific expression of the synthetic cry1C gene is useful for the control of S. litura which was rather resistant to the other types of Bt toxins.

Amino Acid Sequence↗
PubMed2001

Protein engineering of novel proteinase inhibitors and their effects on the growth of Spodoptera exigua larvae.

Novel types of proteinase inhibitors with multi-inhibitory activities were generated by replacement of phytocystatin domains in sunflower multi-cystatin (SMC) by the serine proteinase inhibitor BGIT from bitter gourd seeds. Two chimeric inhibitors SMC-T3 and SMC-T23, in which the third domain in SMC and the second and third domains in SMC were replaced by BGIT, acquired trypsin inhibitory activity (Ki: 1.46 x 10(-7) M and 1.75 x 10(-7) M), retaining inhibitory activity toward papain (Ki: 4.5 x 10(-8) M and 1.52 x 10(-7) M), respectively. We compared the chimeric inhibitors and the recombinant SMC (r-SMC) in relation to their effects on the growth of larval Spodoptera exigua. When the second instar larvae were reared on a diet containing rSMC, SMC-T3, or SMC-T23 for ten days, a significant reduction in weight gain was observed. Mean weights for rSMC, SMC-T3, and SMC-T23 were 43 mg, 32 mg, and 43 mg, respectively, as compared with that (60 mg) for the absence of the inhibitor. In contrast, BGIT had little effect on the growth of the S. exigua larvae. This result indicated that the chimeric inhibitor SMC-T3 with two phytocystatin domains and one serine proteinase inhibitor domain is an efficient inhibitor of proteinases in the S. exigua larvae. Therefore, this novel type of proteinase inhibitor with multi-inhibitory activities may represent a promising protein for successful application to a transgenic plant with insect resistance.

Animals↗
PubMed2000

Adaptation of an insect cell line of Spodoptera frugiperda to grow at 37 degrees C: characterization of an endodiploid clone.

Sf21 and Sf9 cell lines established from the lepidoptera Spodoptera frugiperda do not display major induction of heat shock proteins when exposed to a temperature of 37 degrees C. After some months of adaptation at 37 degrees C we obtained two new cell lines, Sf21-HT and Sf9-HT, which have now been established for several years in our laboratory. The Sf9-HT line displays a slightly shorter doubling time at 37 degrees C than the wild type at 28 degrees C, but cell lethality gives rise to an earlier growth arrest. The composition of total lipid extract from heat-adapted cells reveals a higher sphingomyelin to phosphatidylcholine ratio and a higher percentage of saturated fatty acids, which are expected for the lower membrane fluidity, required for thermotolerance. The cell volume of Sf9-HT is doubled, and by flow cytometry we showed that the DNA content is twice that in the parental cell line. Karyotypic examination of metaphasic cells achieved under epifluorescence microscopy revealed a doubled chromosome number in Sf9-HT.

Animals↗
PubMedDate not supplied

Establishment of phagocytic cell lines from larval hemocytes of the beet armyworm, Spodoptera exigua.

Hemolymph was taken from beet armyworm (Spodoptera exigua) larvae and a new hemocyte cell line (SeHe920-1a) was established by supplementing the culture medium with a reduced form of glutathione to avoid the activation of prophenoloxidase cascade. To evaluate the phagocytic ability of the SeHe920-1a cells, polystyrene microspheres of two sizes (6.14 +/- 0.45 microm and 2.84 +/- 0.14 microm in diameter) and inactivated spores of an entomopathogenic microsporidium, Vairimorpha sp. NIS M12 (5.10 +/- 0.21 microm x 2.00 +/- 0.11 microm), were introduced into the cell culture. The SeHe920-1a cells had higher phagocytic ability than other lepidopteran cell lines that were not derived from the hemocytes. When microsporidian spores were inoculated, 27% of SeHe920-1a cells were observed to take up spores (average 1.7 spores per cell). By cloning SeHe920-1a cells, 12 cell lines were established and designated SeHe920Y1 to SeHe920Y12. In comparison with the parental cell line, phagocytic activity was enhanced in SeHe920Y6, SeHe920Y10, and SeHe920Y11 cell lines and especially in the SeHe920Y7 cell line, where approximately 50% of cells were phagocytic and the average number of microsporidian spores engulfed per cell was twice that of the SeHe920-1a cell line.

Animals↗
PubMedDate not supplied

Action of major insecticide groups on insect cell lines of the beet armyworm, Spodoptera exigua, compared with larvicidal toxicity.

In a first series of experiments, the biological response of a continuous cell line of the beet armyworm, Spodoptera exigua, was tested with different groups of insecticides with different modes of actions: acetylcholinesterase inhibitors, acetycholine receptor agonists, inhibitors and uncouplers of oxidative phosphorylation, site I electron transport inhibitors, gamma-aminobutyric acid receptor inhibitors, chitin synthesis inhibitors, and juvenile hormone analogues. From the concentration response curves, 50% inhibition concentration (IC(50)) values were calculated. The most active compound in vitro was pyridaben with an IC(50) value of 0.0083 ppm. In a second series of experiments, the toxicity of these insecticide groups was determined on third-instar larvae of S. exigua, and lethal concentration with 50% kill (LC(50)) values were used in the evaluation of their in vivo biological activity. Toxicity bioassays showed that lufenuron was the most toxic (LC(50) = 0.098 ppm). To explain the discrepancies in biological responses in vitro with insect cells compared with in vivo conditions with whole third-instar larvae, the significance of different detoxifying enzyme systems was tested. P(450) monooxygenases, esterases, and glutathione S-transferases were measured in third-instar larvae and cells of S. exigua. Data are discussed in terms of the usefulness of insect cell cultures as tools in the screening for novel insecticide actions.

Animals↗
PubMedDate not supplied

Phenotypic and genetic diversity of Bacillus thuringiensis strains isolated in India active against Spodoptera litura.

Bacillus thuringiensis strains isolated from different agroclimatic regions of India were found to harbor cry1 family genes. Of 831 strains 18 that were found to produce 130- and 68-kDa mol wt proteins in sodium dodecyl sulfate polyacry1amide gel electrophoresis were subjected to bioassay against second instar larvae of Spodoptera litura. According to the time response curve, while the highest toxic activity against S. litura was observed in PBT-782 with an LT50 of 25.46 h, strains PBT-372, PBT-574, PBT-801, and PBT-716 in descending order of merit had LT50 values of 36.81, 48.18, 50.35, and 73.53 h. The results of the field experiment testing the efficacy of different B. thuringiensis strains in controlling S. litura larvae infecting peanut plants showed that the chemical insecticide chlorpyriphos was the most effective in controlling S. litura throughout the study period. However, among B. thuringiensis strains, PBT-372 was superior. All the B. thuringiensis strains except PBT-689 were found to contain cry1Ac1-type gene. However, only nine strains contained cry1Aa1 gene. While cry1Ab1 was present only in PBT-372 and PBT-689, cry1Ca1 was present in PBT-574, PBT-688, PBT-689, and PBT-695. cry1Da1 was detected only in PBT-688 and PBT-692. None of the strains contained cry1Ba1 and cry1Ea1 genes. When polymerase chain reaction analysis using cry1Ca1 primer was performed, PBT-695 produced an unexpected 739-bp product, which showed 33% homology with cry1Ca1 gene between nucleotides 1819 and 2107. Our results indicated that among the field-collected B. thuringiensis strains, PBT-372 harbors multiple cry-type genes and could be employed for biological control of insects.

Animals↗
PubMed1999

Alteration of glucose consumption kinetics with progression of baculovirus infection in Spodoptera frugiperda cells.

We have used the initial-rate approach to characterize changes in the glucose consumption kinetics of baculovirus-infected Spodoptera frugiperda clone 9 (Sf9) cells with the progression of the infection process. The specific glucose consumption rate (qG) of cultured baculovirus-infected Sf9 cells was measured at 4, 8, 12, 16, and 24 h postinfection (h.p.i.) in media containing 4-35 mM glucose. Higher medium glucose concentrations resulted in higher final extracellular virus and recombinant beta-galactosidase yields. qG was related to the extracellular glucose concentration by means of a Michaelis-Menten relationship. The apparent Michaelis-Menten constant (K(m)) for glucose consumption was found not to change significantly during the progression of the infection process, and remained between 6.2 and 7.2 mM. However, the maximal specific glucose consumption rate (qGmax) was found to rapidly increase after infection, peaking at 16 h.p.i. at a value four times that for uninfected Sf9 cells. The kinetic analysis of glucose consumption rates in baculovirus-infected Sf9 cells presented here will aid in the optimal design and operation of bioreactor systems for the large-scale production of recombinant products from the baculovirus/insect cell system.

Animals↗
PubMed2000

Comparison of Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Spodoptera frugiperda, and COS7 cells for recombinant gene expression. Application to a rabbit liver carboxylesterase.

Expression of a rabbit liver carboxylesterase has been achieved in several different model systems including Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Spodoptera frugiperda, and COS7 cells. Although, recombinant protein was observed in E. coli sonicates, little or no enzymatic activity was detected. Similarly, no activity was observed following expression in S. cerevisiae. In contrast, active protein was produced in P. pastoris, from S. frugiperda following baculoviral infection and in COS7 cells following transient transfection of plasmid DNA. For the preparation of small amounts of protein for kinetic and biochemical studies, enzyme expressed in P. pastoris has proved sufficient. However, to produce large amounts of carboxylesterase for structural studies, baculoviral-mediated expression of a secreted form of the protein in S. frugiperda was the most efficient. Using this system, we have generated and purified milligram quantities of essentially pure protein. These results demonstrate that the choice of in vitro system for the generation of large amounts of active carboxylesterase, and probably most endoplasmic reticulum processed proteins, is crucial for high level expression and subsequent purification.

Animals↗
PubMedDate not supplied

Insect growth regulatory activity of some extracts and compounds from Parthenium argentatum on fall armyworm Spodoptera frugiperda.

The methanolic extract from aerial parts of Parthenium argentatum, afforded argentatin A and B. These compounds were evaluated for their effect on the fall armyworm (Spodoptera frugiperda). Toosendanin, a commercial insecticide derived from Melia azedarach was used as positive control. When tested for activity, using neonate larvae into the no-choice artificial diet bioassays, argentatin A, argentatin B and methanol extract caused significant growth inhibitory activity with GC50 of 17.8, 36.1 and 6.4 ppm at 7 days, respectively, and increased the development time of surviving larvae in a concentration-dependent manner with RGI values of 0.40, 0.60 and 0.26, at 25.0, 25.0 and 5.0 ppm, respectively. In addition, it was possible to observe in most of the treated groups a significant delay in the time of pupation, adult emergence and deformities. Acute toxicity against adults of S. frugiperda was also found, MeOH extract had the most potent activity with LD50 value of 3.10 ppm. In addition, MeOH extract and argentatin A caused acetylcholinesterase inhibition of 93.7% and 90.0%, at 5.0 and 50.0 ppm, respectively; whereas argentatin B had only slight inhibitory activity. Therefore, the MeOH extract was identified as insecticidal extract from P. argentatum with activity at concentrations above 15.0 ppm.

Acetylcholinesterase↗
PubMedDate not supplied

Acetylcholinesterase and insect growth inhibitory activities of Gutierrezia microcephala on fall armyworm Spodoptera frugiperda J.E. Smith.

From the aerial parts of Gutierrezia microcephala (Asteraceae), four oxyflavones were isolated, namely 5,7,2'-trihydroxy-3,6,8,4',5'-pentamethoxyflavone (1); 5,7,4'-trihydroxy-3,6,8-trimethoxyflavone (2); 5,7,2',4'-tetrahydroxy-3,6,8,5'-tetramethoxyflavone (3); 5,2'-dihydroxy-3,6,7,8,4',5'-hexamethoxyflavone (4), and an ent-clerodane, bacchabolivic acid (5). Compounds 1-5, the synthetic methyl ester (6), n-hexane and MeOH extracts were evaluated against the fall armyworm (Spodoptera frugiperda). Gedunin, a known insect growth regulator isolated from Cedrela spp. was used as a positive control. When tested for activity on neonate larvae into the no-choice artificial diet bioassay, flavone (1), clerodane (5), its methyl ester (6), MeOH and n-hexane extracts caused significant larval mortality with MC50 of 3.9, 10.7, 3.46, 7.95 and 7.5 ppm at 7 days, respectively, as well as growth reduction. They also increased the development time of surviving larvae and a significant delay in time to pupation and adult emergence. Acute toxicity against adults of S. frugiperda was also found, 5, 6, gedunin and n-hexane extract had the most potent activity with LD50 value of 6.59, 15.05, 10.78, and 12.79 ppm, respectively. In addition, MeOH, n-hexane extracts, 5, 6 and gedunin caused acetylcholinesterase inhibition with 93.7, 100, 90.2, 62.0 and 100% at 50.0 ppm, respectively; whereas 1-4 exhibited only moderate inhibitory activity. Compounds 1, 5 and 6 showed inhibitory activities comparable with gedunin. These compounds could be responsible of the insect growth inhibitory activity of this plant.

Acetylcholinesterase↗