Specific Incorporation of L-Glutamine into Volicitin in the Regurgitant of Spodoptera litura

Volicitin, [N-(17-hydroxylinolenoyl)-L-glutamine], was identified as an elicitor of plant volatiles from a Spodoptera exigua regurgitant. It has been proposed that gut microbes synthesize volicitin from glutamine, a predominant amino acid component in the insect gut. However, we found that glutamine was not a major component in the regurgitant of Spodoptera litura, although L-glutamine was exclusively incorporated into volicitin by S. litura fed on diets enriched with various amino acids. This selectivity of glutamine as a substrate was not due to a dominant occurrence in the insect gut.     

In vitro biosynthesis of volicitin in Spodoptera litura

Volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] and N-linolenoyl-L-glutamine, originally identified in the regurgitant of Spodoptera exigua, induce damaged corn leaves to release volatile compounds which enable parasitic wasps to locate host caterpillars. Here we demonstrate the in vitro biosynthesis of volicitin for the first time by using gut tissues of Spodoptera litura larvae, as well as N-linolenoyl-L-glutamine. When crop, midgut tissues, peritrophic membrane and gut contents isolated from S. litura were incubated with sodium linolenate and L-[alpha-15N] glutamine, not only 15N-labeled N-linolenoyl-L-glutamine but 15N-labeled volicitin was detected mainly in the midgut incubation by LCMS and LCMSMS analysis. In contrast, there were negligible amounts of the newly biosynthesized compounds in the gut content incubation. Furthermore, the microsomal fraction obtained from the gut tissues clearly showed specific incorporation of glutamine. This substrate selectivity accounts for the exclusive uptake of glutamine by fatty acid amides (FAAs) in the noctuid caterpillars, even though glutamine was not a major component in the regurgitant. Additionally, intensive chemical analyses revealed that more than 20% of glutamine in hemolymph was present as conjugates in gut contents. These results suggest that FAA compounds are actively synthesized by caterpillar tissues and might play important physiological role(s) in glutamine metabolism.     

Efficient incorporation of unsaturated fatty acids into volicitin-related compounds in Spodoptera litura (Lepidoptera: Noctuidae)

We introduced efficient incorporation of unsaturated fatty acids into volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] and N-linolenoyl-L-glutamine, insect-derived elicitors of plant volatiles, in the common cutworms Spodoptera litura by the incubation of larval gut tissues with unsaturated (linolenic, linoleic, and oleic acids) or saturated fatty acids (palmitic and stearic acids) sodium salt, and L-[alpha-(15)N]glutamine.     

In situ translocation of volicitin by beet armyworm larvae to maize and systemic immobility of the herbivore elicitor <Emphasis Type="Italic">in planta</Emphasis>

Volicitin (N-[17-hydroxylinolenoyl]-l glutamine) present in the regurgitant of beet armyworm (Spodoptera exigua) activates the emissions of volatile organic compounds (VOCs) when in contact with damaged corn (Zea mays L.) leaves. VOC emission in turn serves as a signaling defense for the plant by attracting female parasitic wasps that prey on herbivore larvae. Chemical tracking of volicitin within plants has yet to be reported. Here we present biochemical data that beet armyworm regurgitant serves as a vector for the introduction of volicitin to the site of leaf damage under natural feeding conditions. Corn seedlings were ¹⁴CO₂-labeled in situ, and beet armyworm larvae were allowed to feed on the labeled leaves. Herbivore oral secretions collected from late-third-instar larvae contained approximately 120 pmol volicitin (0.05 nCi pmol^–1) per larva. When radiochemically labeled larvae were placed on unlabeled leaves, the amount of volicitin introduced to the damaged site was approximately 5.0 nCi (calc. 100 pmol/larvae). The mobility of volicitin in leaves was examined by allowing radiolabeled beet armyworms to feed on unlabeled plants. In such tracking experiments, radioactivity was not detected in the upper leaves; however, the exogenous application of 5 nCi of [U-¹⁴C]sucrose to the lower leaf did result in subsequent radioactivity being detected in the upper portion of the plant. The detection of labeled sucrose with the same radioactivity as that of administered volicitin indicated that volicitin was not readily transported to undamaged leaves and that volicitin may not directly serve as a mobile messenger in triggering the emissions of VOCs systemically.Abbreviations BAW Beet armyworm (Spodoptera exigua) - dpm Disintegrations per minute - FAA Fatty acid amide - JA Jasmonic acid - VOC Volatile organic compound     

斜纹夜蛾幼虫肠道细菌分离鉴定及其功能初步分析

昆虫肠道微生物对其寄主的生长发育、营养代谢、免疫以及农药抗性等方面都发挥着重要作用。为研究斜纹夜蛾Spodoptera litura幼虫肠道细菌的多样性,并为其功能验证做准备,本文利用传统微生物分离纯培养方法从斜纹夜蛾4龄幼虫肠道中共分离鉴定得到10株细菌,分别为属于变形菌门(Proteobacteria)的脱氮假单胞菌(Pseudomonas denitrificans),不动细菌(Acinetobacter sp.),肺炎克雷伯氏菌(Klebsiella pneumoniae)和肠杆菌(Enterobacter sp.);属于厚壁菌门(Firmicutes)的鸡葡萄球菌(Staphylococcus gallinarum),蒙氏肠球菌(Enterococcus mundtii),蜡样芽胞杆菌(Bacillus cereus)和枯草芽胞杆菌(Bacillus subtilis)以及放线菌门(Actinobacteria)的微杆菌(Microbacteriums sp.)和乳酪棒杆菌(Corynebacterium casei)。变形菌门和厚壁菌门是斜纹夜蛾肠道可培养细菌中的优势菌群。功能验证实验表明肠杆菌具备纤维素降解能力,微杆菌具备很强的苯酚降解能力。本研究为未来深入研究斜纹夜蛾肠道微生物的功能提供了方向和菌株材料。     

Absolute configuration of volicitin from the regurgitant of lepidopteran caterpillars and biological activity of volicitin-related compounds

Volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] and N-linolenoyl-L-glutamine are known as insect-produced plant volatile elicitors. The absolute configuration of the hydroxylinolenoyl moiety of volicitin from three noctuid species, Helicoverpa armigera, Mythimna separata and Spodoptera litura, was determined to be all 17S in high enantiomeric excess. When treated with 30 pmol of (17S)- and (17R)-volicitin, corn seedlings were induced to release volatiles, there being no significant difference in the amount released between the two isomers. On the other hand, N-linolenoyl-L-glutamine was only about 30% as active as volicitin. Among several synthesized N-linolenoylamino acid conjugates, only the L-glutamine conjugate induced the emission of volatile organic compounds. These results show that the L-glutamine moiety of volicitin played a more critical role than the hydroxyl moiety, although both moieties affected the elicitor activity inducing the release of volatiles.     

Effects of feeding Spodoptera littoralis on lima bean leaves. I. Membrane potentials, intracellular calcium variations, oral secretions, and regurgitate components

Membrane potentials (V(m)) and intracellular calcium variations were studied in Lima bean (Phaseolus lunatus) leaves when the Mediterranean climbing cutworm (Spodoptera littoralis) was attacking the plants. In addition to the effect of the feeding insect the impact of several N-acyl Glns (volicitin, N-palmitoyl-Gln, N-linolenoyl-Gln) from the larval oral secretion was studied. The results showed that the early events upon herbivore attack were: a) a strong V(m) depolarization at the bite zone and an isotropic wave of V(m) depolarization spreading throughout the entire attacked leaf; b) a V(m) depolarization observed for the regurgitant but not with volicitin [N-(17-hydroxy-linolenoyl)-Gln] alone; c) an enhanced influx of Ca(2+) at the very edge of the bite, which is halved, if the Ca(2+) channel blocker Verapamil is used. Furthermore, the dose-dependence effects of N-acyl Gln conjugates-triggered influx of Ca(2+) studied in transgenic aequorin-expressing soybean (Glycine max) cells, showed: a) a concentration-dependent influx of Ca(2+); b) a configuration-independent effect concerning the stereochemistry of the amino acid moiety; c) a slightly reduced influx of Ca(2+) after modification of the fatty acid backbone by functionalization with oxygen and; d) a comparable effect with the detergent SDS. Finally, the herbivore wounding causes a response in the plant cells that cannot be mimicked by mechanical wounding. The involvement of Ca(2+) in signaling after herbivore wounding is discussed.     

Bioinsecticidal activity of Murraya koenigii miraculin-like protein against Helicoverpa armigera and Spodoptera litura

Miraculin-like proteins, belonging to the Kunitz superfamily, are natural plant defense agents against pests and predators, and therefore are potential biopesticides for incorporation into pest-resistant crops. Here, a miraculin-like protein from Murraya koenigii was assessed for its in vitro and in vivo effects against two polyphagous lepidopteran insect pests, Helicoverpa armigera and Spodoptera litura. M. koenigii miraculin-like protein (MKMLP) inhibited the trypsin-like activity and total protease activity of H. armigera gut proteinases (HGP) by 78.5 and 40%, respectively, and S.litura gut proteinases (SGP) by 81 and 48%, respectively. The inhibitor was stable and actively inhibited the proteolysis of both HGP and SGP enzymes for up to 72 h. Incorporation of MKMLP into artificial diet adversely affected the growth and development of pests in a dose-dependent manner. After 10 days of feeding on diets containing 200 μM MKMLP, larval weight was reduced to 69 and 44.8% and larval mortality was increased to 40 and 43.3% for H. armigera and S litura, respectively. The LC(50) of MKMLP was 0.34 and 0.22% of the diet for H.armigera and S. litura, respectively. These results demonstrate the efficacy of MKMLP as a potential plant defense agent against H. armigera and S. litura.     

Insecticidal activity and processing in larval gut juices of genetically engineered 130-kDa proteins of Bacillus thuringiensis subsp. aizawai.

The 130-kDa insecticidal protein (IP) of Bacillus thuringiensis subsp. aizawai is proteolytically processed in the gut juice of susceptible insect larvae to yield an insecticidally active 60-kDa fragment. Twenty-seven mutant IP genes with the replacement of codons for Arg and Lys with codons for Gln in the active fragment and its adjacent regions of the 130-kDa IP were constructed by site-directed mutagenesis and expressed in Escherichia coli cells. The produced mutant IPs at Arg87, Arg131, Arg198, Arg311, Arg368, Arg402, Arg458, Arg502, Arg512, Arg524, Arg526, Arg528, and Arg601 had reduced insecticidal activity against Spodoptera litura larvae. The mutant at Arg601 was sensitive to proteolytic digestion in the gut juice of S. litura larvae. Although the mutants at Arg619, Lys622, and Lys637 had nearly the same activity as that of the wild type, the mutant with the triple replacement at Arg619, Lys622, and Lys637 was 2.5 times more active against S. litura larvae than the wild type. This triple mutant showed a slightly different processing profile in the gut juice than that of the wild type.     

Gut bacteria may be involved in interactions between plants, herbivores and their predators: microbial biosynthesis of N-acylglutamine surfactants as elicitors of plant volatiles

N-Acylamino acids are dominant and widespread constituents of insect oral secretions (regurgitants), serving the insect as biosurfactants in the digestive process. During feeding the conjugates may be introduced into damaged leaves and contribute there to the elicitation of plant defenses such as the induction of volatile biosynthesis. From gut segments of Spodoptera exigua, Mamestra brassicae and Agrotis segetum 23 bacterial strains were isolated, ten of which were able to synthesise typical lepidopteran N-acylamino acids from externally added precursors. Four strains, Providencia rettgeri, Ochrobactrum spec., Myroides odoratus and Acinetobacter sp. genospecies 11 were identified on the basis of their 16 S rDNA. The organisms displayed a very broad substrate tolerance, since fatty acids of different chain length and different degree of saturation were converted into N-acylamino acids. Moreover, most of the proteinogenic amino acids, but not glutamic and aspartic acid, were used as substrates. The dominant occurrence of fatty acids conjugated with glutamine may result from a preferred transport of glutamine from the hemolymph into the gut of the insects. The involvement of bacteria in the biosynthesis of compounds which play a pivotal role in the interaction of plants, herbivores and their predators adds a new trophic level to this complex network of interactions. Due to their short generation cycle and the ease of adaptation endosymbiontic bacteria may have an outstanding importance for the coevolution of plant-insect interactions.     

Nucleotide sequence and molecular characterization of the structural glycoprotein gp41 gene homologue of Spodoptera litura nucleopolyhedrosis virus (spltNPV-I)

The structural glycoprotein gene gp41 homologue of Spodoptera litura nucleopolyhedrosis virus (SpltNPV-I *) was identified in the 4.0 kb EcoRI-L fragment of the viral genome. The nucleotide sequence of 2063 bp of this fragment revealed an open reading frame of 1014 nucleotides to encode a polypeptide of 337 amino acids. Analysis of nucleotide and deduced amino acid sequences of the putative ORF indicated its identity with gp41 protein of other baculoviruses sharing maximum homology with that of Spodoptera frugiperda nucleopolyhedrosis virus (SfNPV). The coding sequence was preceded by an AT-rich region containing the consensus baculoviral late promoter motif RTAAG. The putative SpltNPV gp41 ORF was abundantly expressed as a 37 kDa apoprotein in E. coli and as a 50 kDa glycoprotein in Sf9 cells. The recombinant protein expressed in insect cells was glycosylated (20%) and has GlcNAc as the terminal sugar. The gene is conserved among baculoviruses and places SpltNPV-I close to Spodoptera frugiperda and Spodoptera exigua NPVs in phylogenetic tree.     

Synergistic interactions between volicitin,jasmonic acid and ethylene mediate insect-induced volatile emission in Zea mays

Plants display differential responses following mechanical damage and insect herbivory. Both caterpillar attack and the application of caterpillar oral secretions (OS) to wounded leaves stimulates volatile emission above mechanical damage alone. Volicitin ( N- 17-hydroxylinolenoyl- l -glutamine), present in beet armyworm (BAW, Spodoptera exigua ) OS, is a powerful elicitor of volatiles in excised maize seedlings ( Zea mays cv. Delprim). We consider some of the mechanistic differences between wounding and insect herbivory in maize by examining the activity of volicitin, changes in jasmonic acid (JA) levels, and volatile emission from both intact plant and excised leaf bioassays. Compared to mechanical damage alone, volicitin stimulated increases in both JA levels and sesquiterpene volatiles when applied to intact plants. In a bioassay comparison, excised leaves were more sensitive and produced far greater volatile responses than intact plants following applications of both volicitin and JA. In the excised leaf bioassay, volicitin applications (10–500 pmol) to wounded leaves resulted in dose dependent JA increases and a direct positive relationship between JA and sesquiterpene volatile emission. Interestingly, volicitin-induced JA levels did not differ between intact and excised bioassays, suggesting a possible interaction of JA with other regulatory signals in excised plants. In addition to JA, insect herbivory is known to stimulate the production of ethylene. Significant increases in ethylene were induced only by BAW herbivory and not by either wounding or volicitin treatments. Using intact plant bioassays, ethylene (at 1 µl l⁻¹ or less) greatly promoted volatile emission induced by volicitin and JA but not mechanical damage alone. For intact plants, wounding, elicitor-induced JA and insect-induced ethylene appear to be important interacting components in the stimulation of insect-induced volatile emission.     

Action of the Protease from Streptomyces cellulosae on l-Leu-Gly-Gly

We introduced efficient incorporation of unsaturated fatty acids into volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] and N-linolenoyl-L-glutamine, insect-derived elicitors of plant volatiles, in the common cutworms Spodoptera litura by the incubation of larval gut tissues with unsaturated (linolenic, linoleic, and oleic acids) or saturated fatty acids (palmitic and stearic acids) sodium salt, and L-[α-¹⁵N]glutamine.     

Bitter gourd proteinase inhibitors: potential growth inhibitors of Helicoverpa armigera and Spodoptera litura

Proteinase inhibitors (PIs) from the seeds of bitter gourd (Momordica charantia L.) were identified as strong inhibitors of Helicoverpa armigera gut proteinases (HGP). Biochemical investigations showed that bitter gourd PIs (BGPIs) inhibited more than 80% HGP activity. Electrophoretic analysis revealed the presence of two major proteins (BGPI-1 and-2) and two minor proteins (BGPI-3 and-4) having inhibitory activity against both trypsin and HGP. The major isoforms BGPI-1 and BGPI-2 have molecular mass of 3.5 and 3.0 kDa, respectively. BGPIs inhibited HGP activity of larvae fed on different host plants, on artificial diet with or without added PIs and proteinases excreted in fecal matter. Degradation of BGPI-1 by HGP showed direct correlation with accumulation of BGPI-2-like peptide, which remained stable and active against high concentrations of HGP up to 3 h. Chemical inhibitors of serine proteinases offered partial protection to BGPI-1 from degradation by HGP, suggesting that trypsin and chymotrypsin like proteinases are involved in degradation of BGPI-1. In larval feeding studies, BGPIs were found to retard growth and development of two lepidopteran pests namely Helicoverpa armigera and Spodoptera litura. This is the first report showing that BGPIs mediated inhibition of insect gut proteinases directly affects fertility and fecundity of both H. armigera and S. litura. The results advocate use of BGPIs to introduce insect resistance in otherwise susceptible plants.     

昆虫杆状病毒泛素基因研究进展

昆虫杆状病毒是目前已知唯一编码泛素(ubiquitin)的病毒。迄今,已克隆了8种该类病毒的泛素基因。与真核生物Uba52(80)相似,这些基因在一个泛素分子的C末端都有不同长度的融合,其中斜纹夜蛾核多角体病毒(Spodopteralituramulticapsidnucleopolyhedrovirus,SpltMNPV)的ubiquitingp37基因是一个典型的融合基因。近年来,对苜蓿银纹夜蛾核多角体病毒(Autographacaliforniamulticapsidnucleopolyhedrovirus,AcMNPV)泛素的定位与功能研究取得了重要进展 。     

Isolation, gene expression and solution structure of a novel moricin analogue, antibacterial peptide from a lepidopteran insect, Spodoptera litura

An antibacterial peptide was isolated from a lepidopteran insect, Spodoptera litura. The molecular mass of this peptide was determined to be 4489.55 by matrix assisted laser desorption/ionization-time of flight mass (MALDI-TOF MS) spectrometry. The peptide consists of 42 amino acids and the sequence has 69-98% identity to those of moricin-related peptides, antibacterial peptides from lepidopetran insects. Thus, the peptide was designated S. litura (Sl) moricin. Sl moricin showed a broad antibacterial spectrum against Gram-positive and negative bacteria. Sl moricin gene was inducible by bacterial injection and expressed tissue-specifically in the fat body and hemocytes. Furthermore, the solution structure of Sl moricin was determined by two-dimensional (2D) 1H-nuclear magnetic resonance (NMR) spectroscopy and hybrid distance geometry-simulated annealing calculation. The tertiary structure revealed a long alpha-helix containing eight turns along nearly the full length of the peptide like that of moricin, confirming that Sl moricin is a new moricin-like antibacterial peptide. These results suggest that moricin is present not only in B. mori but also in other lepidopteran insects forming a gene family.     

Purification of Lysozyme from Hemolymph of Tobacco Cutworm, Spodoptera litura

ABSTRACT Lysozyme is one of the components of the innate immunity of the insects. The lysozyme has been isolated by heat treatment, cation exchange, and reversed-phase chromatography from immunized hemolymph of last instar larvae of Spodoptera litura. The hemolymph immunized with the insect nonpathogen, Escherichia coli was collected 2 days after the abdominal injection. The molecular weight of Spodoptera lysozyme was estimated to be about 15 kDa by SDS-PAGE and it is great similarity with Agrius lysozyme, which recently purified from larval hemolymph of Agrius convolvuli.     

Identification of volicitin-related compounds from the regurgitant of lepidopteran caterpillars

Volicitin-related compounds were found in the oral secretion of the three noctuid species, Helicoverpa armigera, Mythimna separata and Spodoptera litura, and one sphingid species, Agrius convolvuli. Volicitin [N-(17-hydroxylinolenoyl)-L-glutamine], N-(17-hydroxylinoleoyl)-glutamine, N-linolenoylglutamine and N-linoleoylglutamine were identified in the secretion from the noctuid larvae. In secretions from the sphingid larvae, N-linolenoylglutamine and N-linoleoylglutamine were the main components. Furthermore, there were significant differences in the amounts of the N-acylamino acid conjugates in the secretions from the three noctuid species. These results suggest that the proportion of volicitin-related compounds in the regurgitant was species-specific.     

斜纹夜蛾核型多角体病毒分子生物学研究进展

斜纹夜蛾核型多角体病毒(Spodoptera lituramulticapsid nucleopolyhedrovirus,SpltMNPV)属NPV科A亚群。近几年有关该病毒的序列测定、基因结构、功能和表达调控等系统的分子生物学研究工作进展迅速,特别是对一些重要基因的结构分析,有助于筛选毒力较强的杀虫毒株,并为这一病毒杀虫剂的改良和发展以及组建昆虫杆状病毒表达载体奠定基础。综述了与SpltMNPV相关的分子生物学领域的研究进展。