alpha-Amanitin-Resistant Viral RNA Synthesis in Nuclei Isolated from Nuclear Polyhedrosis Virus-Infected Heliothis zea Larvae and Spodoptera frugiperda Cells

We performed three types of experiments to test the hypothesis that abortive infection of T5 bacteriophage in Escherichia coli (ColIb+) is due to internally released colicin. (i) We measured the sensitivity of cells to colicin under a variety of conditions and then looked at the plating efficiency of T5 in ColIb+ cells under these same conditions. Cells grown at 42 degrees C or with hexanol had a reduced sensitivity to externally added colicin and an increased efficiency for T5 when the ColIb plasmid was present in the infected cells. Phage growth was far from normal, however. (ii) We measured the colicin sensitivity of a mutant bacterium that grew T5 normally even in the presence of the ColIb plasmid and measured the plating efficiency of T5 on another mutant that was colicin tolerant. Here again, the correlation between colicin activity and inhibition of phage replication was not complete. (iii) We looked for colicin-negative plasmid mutants and tested the ability of cells containing these plasmids to support the growth of T5. These experiments used Tn5, a kanamycin resistance transposon, as the mutagen. All possible combinations of colicin production and phage inhibition were found, including mutants that produced no colicin but still inhibited phage production.     

Temporal oviposition patterns of Heliothis zea and Spodoptera ornithogalli

Egg production under laboratory conditions was examined over the lifespan of Heliothis zea (Boddie) and Spodoptera ornithogalli Guenée (Lepidoptera: Noctuidae). Although H. zea oviposits singly and S. ornithogalli oviposits in masses, temporal trends were similar. Egg numbers peaked shortly after mating and then rapidly declined. Egg weights also peaked shortly after mating, but decreased gradually over time. Temporal oviposition patterns were more erratic for unmated than mated females, suggesting the importance of mating in establishing the shape of the oviposition curve.

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Résumé La production d'ufs d'Heliothis zea (Boddie) et de Spodoptera ornithogalli Guenée (Lepidoptera: Noctuidae) durant toute la vie adulte a été examinée au laboratoire. Bien que H. zea dépose ses ufs isolément et que S. ornithogalli les dépose en groupes, les courbes temporelles d'oviposition chez des individus fécondés sont similaires. Le nomber d'ufs culmine peu après l'accouplement, puis décline rapidement. Le poids des ufs culmine aussi peu après l'accouplement, puis décroît graduellement. Des femelles fécondées produisent environ deux fois autant d'ufs que les femelles non fécondées, quoique la longévité ne diffère pas significativement entre les deux groupes. Des femelles non fécondées montrent des distributions temporelles plus irrégulières que des femelles fécondées, en ce qui concerne le nombre des ufs et leur poids. Ces irrégularités ont été attribuées à la tendance des femelles non fécondées à retenir leur ufs, ce qui suggère que l'accouplement exerce une influence sur la forme de la courbe temporelle d'oviposition. Des femelles d'H. zea contenant un, deux ou trois spermatophores n'ont pas produit des nombres d'ufs significativement différents.

     

Conformational changes on ligand binding in wild-type and mutants from Spodoptera frugiperda midgut trehalase

Trehalase specifically hydrolyses trehalose into two glucose units and is most important in insects and fungi. Previous evidence suggested that Spodoptera frugiperda midgut trehalase (wild type, WT) has substantial conformational changes on binding different substances. Our goal is to understand this mobility. For this, two deletion mutants were produced, lacking regions supposed to be the cause of mobility [(102 residues from the N-terminus (NT) and this portion plus 31 residues from the C-terminus (NCT)]. Circular dichroism spectra before and after denaturation of the enzymes support the assertion that they are appropriately folded. The overall results show that the removal of 102 or 133 amino acids does not greatly change the interaction with the substrate and competitive inhibitors, but leads to a considerable decrease in kcat/Km values from WT 74,500 M^?1 s^?1 to NT 647 M^?1 s^?1 and NCT 1,044 M^?1 s^?1. Diethyl pyrocarbonate His modification only occurs in wild and truncated trehalases in the presence of some ligands. Looking for changes in folding WT, NT, and NCT were incubated with different compounds in the presence of Sypro Orange, that binds to hydrophobic regions increasing its fluorescence. The dye fluorescence is affected by 2 compounds when WT is present, and at least by 5 compounds when NT or NCT are present, suggesting that conformational changes caused by ligand binding occur only in the vicinity of the active site. These data provide physical evidence in favor of a change in folding around the active site caused by ligand binding, in agreement to prior chemical modification and other kinetic data and challenging the hypothesis that N- and C-terminal are the mobile regions.     

The catalytic and other residues essential for the activity of the midgut trehalase from Spodoptera frugiperda

Trehalase (EC 3.2.1.28) hydrolyzes only α, α′- trehalose and is present in a variety of organisms, but is most important in insects and fungi. Crystallographic data showed that bacterial trehalase has D312 and E496 as the catalytical residues and three Arg residues in the active site. Those residues have homologous in all family 37 trehalases including Spodoptera frugiperda trehalase (D322, E520, R169, R227, R287). To test the role of these residues, mutants of trehalase were produced. All mutants were at least four orders of magnitude less active than wild type trehalase and no structural difference between these mutants and wild type enzyme were discernible by circular dichroism. D322A and E520 pH-activity profile lacked the alkaline arm and the acid arm, respectively, suggesting that D322 is the acid and E520 the basic catalyst. Azide increases E520A activity three times, confirming its action as the basic catalyst. Taking into account the decrease in activity after substitution for alanine residue, the three arginine residues are as important as the catalytical ones to trehalase activity. This clarifies the previous misidentification of an Arg residue as the acid catalyst. As far as we know, this is the first report on the functional identification residues important for trehalase activity.     

Digestive enzymes in midgut cells,endo-and ectoperitrophic contents,and peritrophic membranes of Spodoptera frugiperda (lepidoptera) larvae

In the midgut of Spodoptera frugiperda larvae, subcellular fractionation data suggest that aminopeptidase and part of amylase, carboxypeptidase A, dipeptidase, and trypsin are bound to the microvillar membranes; that major amounts of soluble dipeptidase, cellobiase, and maltase are trapped in the cell glycocalyx; and finally that soluble carboxypeptidase, amylase, and trypsin occur in intracellular vesicles. Most luminal acetylglucosaminidase is soluble and restricted to the ectoperitrophic contents. Aminopeptidase occurs in minor amounts bound to membranes both in the ectoperitrophic contents and incorporated in the peritrophic membrane. Amylase, carboxypeptidase A, and trypsin are found in minor amounts in the ectoperitrophic contents (both soluble and membrane-bound) and in major amounts in the peritrophic membrane with contents. Part of the activities recovered in the last mentioned contents corresponds to enzyme molecules incorporated in the peritrophic membrane. The results suggest that initial digestion is carried out in major amounts by enzymes in the endoperitrophic space and, in minor amounts, by enzymes immobilized in the peritrophic membrane. Intermediate and final digestion occur at the ectoperitrophic space or at the surface of midgut cells. The results also lend support to the hypothesis that amylase and trypsin are derived from membrane-bound forms, are released in soluble form by a microapocrine mechanism, and are partly incorporated into the peritrophic membrane. © 1994 Wiley-Liss, Inc.     

Purification and properties of a beta-glycosidase purified from midgut cells of Spodoptera frugiperda (Lepidoptera) larvae

Two beta-glycosidases (BG) (Mr 47,000 and Mr 50,000) were purified from Spodoptera frugiperda (Lepidoptera: Noctuidae) midguts. These two polypeptides associate or dissociate depending on the medium ionic strength. The Mr 47,000 BG probably has two active sites. One of the putative active sites (cellobiase site) hydrolyses p-nitrophenyl beta-D-glucoside (NPbetaGlu) (79% of the total activity in saturated enzyme), cellobiose, amygdalin and probably also cellotriose, cellotetraose and cellopentaose. The cellobiase site has four subsites for glucose residue binding, as can be deduced from cellodextrin cleavage data. The enzymatic activity in this site is abolished after carbodiimide modification at pH 6.0. Since the inactivation is reduced in the presence of cellobiose, the results suggest the presence of a carboxylate as a catalytic group. The other active site of Mr 47,000 BG (galactosidase site) hydrolyses p-nitrophenyl beta-D-galactoside (NPbetaGal) better than NPbetaGlu, cleaves glucosylceramide and lactose and is unable to act on cellobiose, cellodextrins and amygdalin. This active site is not modified by carbodiimide at pH 6.0. The Mr 47,000 BG N-terminal sequence has high identity to plant beta-glycosidases and to mammalian lactase-phlorizin hydrolase, and contains the QIEGA motif, characteristic of the family of glycosyl hydrolases. The putative physiological role of this enzyme is the digestion of glycolipids (galactosidase site) and di- and oligosaccharides (cellobiase site) derived from hemicelluloses, thus resembling mammalian lactase-phlorizin hydrolase.     

Alpha-galactosidases from the larval midgut of Tenebrio molitor (Coleoptera) and Spodoptera frugiperda (Lepidoptera)

There are three midgut alpha-galactosidases (TG1, TG2, TG3) from Tenebrio molitor larvae that are partially resolved by ion-exchange chromatography. The enzymes have approximately the same pH optimum (5.0), pl value (4.6) and Mr value (46000-49000) as determined by gel filtration or native electrophoresis run in polyacrylamide gels with different concentrations. Substrate specificities and functions were proposed for the major T. molitor midgut alpha-galactosidases (TG2 and TG3) based on chromatographic, carbodiimide inactivation, Tris inhibition, and on substrate competition data. Thus, TG2 would hydrolyse alpha-1,6-galactosaccharides, exemplified by raffinose, whereas TG3 would act on melibiose and apparently also on digalactosyldiglyceride, the most important compound in the thylacoid membranes of chloroplasts. Most galactoside digestion should occur in the lumen of the first two thirds of T. molitor larval midguts, since alpha-galactosidase activity predominates there. Spodoptera frugiperda larvae have three midgut alpha-galactosidases (SG1, SG2, SG3) partially resolved by ion-exchange chromatography. The enzymes have similar pH optimum (5.8), pl value (7.2) and Mr value (46000-52000), and at least the major alpha-galactosidase must have an active carboxyl group in the active site. Based on data similar to those described for T. molitor, SG1 and SG3 should hydrolyse melibiose and SG3 should digest raffinose and, perhaps, also digalactosyldiglyceride. The midgut distribution of alpha-galactosidase activity supports the proposal that alpha-galactosidase digestion occurs at the surface of anterior midgut cells in Spodoptera frugiperda larvae.     

Identification of midgut microvillar proteins from Tenebrio molitor and Spodoptera frugiperda by cDNA library screenings with antibodies

The objective of this study was to identify midgut microvillar proteins in insects appearing earlier (Coleoptera) and later (Lepidoptera) in evolution. For this, cytoskeleton-free midgut microvillar membrane from Spodoptera frugiperda (Lepidoptera) and Tenebrio molitor (Coleoptera) were used to raise antibodies. These were used for screening midgut cDNA expression libraries. Positive clones were sequenced, assembled and searched for similarities with gene/protein databases. The predicted midgut microvillar proteins from T. molitor were: cockroach allergens (unknown function), peritrophins (peritrophic membrane proteins), digestive enzymes (aminopeptidase, alpha-mannosidase) and unknown proteins. Predicted S. frugiperda midgut proteins may be grouped into six classes: (a) proteins involved in protection of midgut (thioredoxin peroxidase, aldehyde dehydrogenase, serpin and juvenile hormone epoxide hydrolase); (b) digestive enzymes (astacin, transporter-like amylase, aminopeptidase, and carboxypeptidase); (c) peritrophins; (d) proteins associated with microapocrine secretion (gelsolin, annexin); (e) membrane-tightly bound-cytoskeleton proteins (fimbrin, calmodulin) and (f) unidentified proteins. The novel approach is compared with others and microvillar function is discussed in the light of the predicted proteins.     

The effect of dietary protein on the growth and digestive physiology of larval Heliothis zea and Spodoptera exigua

We compared the ability of larval H. zea (Boddie) and S. exigua (Hubner) to digest and utilize dietary protein by: (a) determining the ability of different concentrations of dietary protein to support larval growth, and (b) determining the effect of different concentrations of dietary protein on the digestive physiology of the organisms, as measured by in vivo digestion of protein and proteolytic activity. Using an artificial diet containing casein as the primary source of protein, we found that H. zea was able to grow at very low levels of casein (≤0.6%), while optimal growth occurred at 1.2% casein. For S. exigua, dietary casein levels of >0.6% were required for growth, and optimal growth occurred at ≥1.2% casein. However, optimal growth in both species was not correlated with the degree of in vivo digestion of protein. The level of in vivo digestion of protein and tryptic activity in S. exigua was proportional to the concentration of dietary protein (under both acute and chronic exposure), and not the amount of food in the gut, suggesting that enzyme synthesis and/or secretion is controlled by a secretagogue mechanism. H. zea only demonstrated a secretagogue mechanism of control of tryptic activity while under acute exposure to different concentrations of casein; under chronic exposure, tryptic activity was uniform regardless of the concentration of dietary casein. When comparing the two species of noctuid, H. zea, which is the larger of the two species, produced less tryptic activity on a unit weight basis, and also digested less of the available dietary protein than S. exigua. Hence, these closely related organisms are processing dietary protein at different efficiencies.     

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.     

Production of a monoclonal antibody to the arylphorin of Heliothis zea

A species-specific monoclonal antibody was produced to whole plasma of fifth instar larvae of the corn earworm, Heliothis zea (Boddie) (Lepidoptera:Noctuidae). This antibody did not cross-react with proteins from the plasma of any of the other lepidopteran larvae tested, including other Heliothis spp. The antigen recognized by this antibody was characterized and found to be arylphorin, a prominent larval storage protein. This conclusion was based on the electrophoretic as well as immunological characteristics of the antigen. Polyacrylamide gel electrophoresis indicated that the antigen had an apparent native molecular weight of 460,000, and that it was composed of subunits having apparent molecular weights of 76,000. The isoelectric point of the antigen was 5.9, with some microheterogeneity being seen. Western blotting of arylphorin against the monoclonal antibody clearly identified the antigen as arylphorin. This protein was not found in egg homogenates or early instar larval plasma, but it was present in large quantities in pupal homogenates and, at trace levels, in adult homogenates. The efficient production and selection of hybridomas producing antibodies specific to H. zea arylphorin using unfractionated plasma as immunogen illustrates the fact that monoclonal antibody technology can produce highly specific antibodies using crude antigen preparations. We discuss the tradeoffs one must accept when choosing this strategy over one using purified immunogens.     

红彩瑞猎蝽对草地贪夜蛾幼虫的控害能力

为明确红彩瑞猎蝽Rhynocoris fuscipes Fabricius成虫对草地贪夜蛾Spodoptera frugiperda J. E. Smith幼虫的控害潜能,室内条件下研究了不同饥饿时间处理红彩瑞猎蝽对草地贪夜蛾3龄幼虫的捕食功能反应、自身密度干扰效应和搜寻效应。结果表明,饥饿0 h、24 h、48 h、72 h和96 h条件下,红彩瑞猎蝽雌、雄成虫对草地贪夜蛾3龄幼虫的功能反应符合Holling Ⅱ型功能反应方程;饥饿程度对红彩瑞猎蝽24 h内捕食量影响明显,饥饿48 h和72 h处理的红彩瑞猎蝽对草地贪夜蛾具有更旺盛的捕食欲,其捕食速度曲线分别为v=0.9047-0.0592t+0.009t2和v=1.1512-0.0702t+0.0011t2,对草地贪夜蛾的捕食量随着时间的变化模型分别为Na=20/（1+e1.968-0.79t)和Na=20/（1+e1.689-0.93t）。不同饥饿程度红彩瑞猎蝽搜寻效应随猎物密度的增加而逐渐降低,其个体间竞争和干扰作用与天敌自身密度变化呈正比,符合Hassell-Varley模型。结果表明,红彩瑞猎蝽成虫对草地贪夜蛾幼虫有较好的防治潜力。     

Susceptibility of Heliothis zea larvae to Nomuraea rileyi at various temperatures

Laboratory studies were conducted to determine the susceptibility of various larval instars of Heliothis zea to different spore doses of Nomuraea rileyi at constant and variable temperatures. The fungus was most effective at 20° and 25°C, with a mortality of 80% and 71%, respectively. At 15°C the disease progressed very slowly with larval mortality occurring in 12–28 days post-treatment. Conversely, at temperature ranges above 15°C, the mortality of the larvae occurred in 6–12 days. Three different combinations of variable temperatures included 20–30°, 25–30°, and 20–35°C, but mortality did not exceed 46%. Larvae in the third to fifth instars were more susceptible to infection than were those in the first and second instars.     

草地贪夜蛾核型多角体病毒对我国草地贪夜蛾不同地理种群毒力的比较分析

草地贪夜蛾Spodoptera frugiperda(J. E. Smith)入侵我国多个地区,逐渐形成地理种群。在草地贪夜蛾核型多角体病毒Spodoptera frugiperda multiple nucleopolyhedrovirus(SfMNPV)生物农药的生产过程中发现,不同地理来源的宿主对SfMNPV的敏感性和产量存在明显差异,显著影响了SfMNPV的生产效率。为解析其敏感性差异及其产生的原因,本研究首先对云南德宏、广东广州、广西钦州、西藏林芝4个地区草地贪夜蛾种群基因型进行了鉴定,采用生物测定法测试SfMNPV对4龄幼虫的口服毒力,然后,通过向幼虫体内注射草地贪夜蛾核多角体病毒出芽型病毒粒子(budded virions, BVs)的方式,越过口服感染中肠的过程,分析敏感性差异发生的阶段。最后比较了高敏感和低敏感种群中肠肠液pH,并基于16S rDNA测序测定了肠道菌群组成。结果表明,广西种群属于纯合玉米型,其余种群为带有水稻型COI标记的杂合玉米型。广西种群对SfMNPV的口服敏感性最低,西藏种群的敏感性最高,但两者注射BV后死亡率差异无统计学意义,暗示病毒敏感性差异...     

Mode of transmission of nuclear-polyhedrosis virus to progeny of adult Heliothis zea

Late second-instar Heliothis armigera larvae were infected with a granulosis and a nuclear polyhedrosis virus, and all the externally visible symptoms for each virus are described. The effects of the virus infections on the feeding habits of the insects are also described, and it was found that a granulosis infection can prolong the larval period by up to 100%. The larvae continue feeding during this prolonged larval period, and can reach almost double the size and mass of normal larvae.It was further found that each of the viruses displays a distinct set of symptoms which could indicate beyond any doubt which of the two viruses induced death in the host.     

Effect of protein type and quantity on growth and development of larval Heliothis zea and Spodoptera exigua and the endoparasitoid Hyposoter exiguae

Based on the use of casein, soy protein, and corn gluten in artificial diets, we show that larval growth of Heliothis zea (Boddie) and Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae) is significantly reduced both by a deficit and surfeit of dietary protein. The optimal level of protein varies with protein type. Similar protein treatments were also used to examine the influence of host diet on growth and development of the endoparasitoid Hyposoter exiguae (Viereck) (Hymenoptera: Ichneumonidae). Dietary protein regimens reducing host larval growth to the greatest extent are most stressful to the parasitoid, irrespective of protein type. Parasitoid growth appeared to be regulated by the nutritional status of the host. The parasitoid required the host to reach ca 20 mg before it could successfully complete its larval development and pupate. This suggests that host hormones may also be involved in parasitoid development. The implications of these findings for theory of tritrophic interactions are discussed.

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Zusammenfassung Mit Hilfe des Gebrauchs von Kasein, Sojaprotein und Maisgluten in künstlicher Diät zeigen wir, dass das Wachstum der Larven von Heliothis zea (Boddie) und Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae) signifikant reduziert ist durch Defizit oder Überschuss von Protein in der Diät. Der optimale Proteingehalt hängt von der Proteinart ab. Ähnliche Proteindiäten wurden auch benutzt, um den Einfluss von Wirtsdiät auf Wachstum und Entwicklung des Endoparasiten Hyposoter exiguae (Viereck) (Hymenoptera: Icheneumonidae) zu untersuchen. Diejenigen Proteingehaltsstufen, die das Larvenwachstum des Wirts zum grössten Ausmass reduzieren, produzieren den grössten Stress für den Parasiten, unabhängig von der Proteinart. Das Wachstum des Parasiten scheint durch den Ernährungszustand des Wirtes reguliert zu werden. Für die erfolgreiche Larvenentwicklung und Verpuppung des Parasiten war ein Gewicht des Wirtes von ca. 20 mg Voraussetzung. Das deutet auf die Möglichkeit hin, dass Wirtshormone für die Entwicklung des Parasiten eine Rolle spielen können. Schlussfolgerungen aus diesen Ergebnissen für die Theorie tritrophischer Wechselbeziehungen werden diskutiert.

     

Toxicity of pyrethroids and effect of synergists to larval and adult Helicoverpa zea, Spodoptera frugiperda, and Agrotis ipsilon (Lepidoptera: Noctuidae)

Based on 48 h LD50 estimates from topical bioassays, cypermethrin was more toxic than permethrin to Helicoverpa zea (Boddie) larvae and adults; however, the two pyrethroids did not differ significantly in their relative toxicities to Spodoptera frugiperda (J. E. Smith) and Agrotis ipsilon (Hufnagle). Larvae of each species generally were more susceptible to cypermethrin and permethrin than respective adults. The only exception to this generalization occurred with H. zea where slight overlap of the 95% confidence intervals with larvae and adult males was observed with cypermethrin. Respective males and females of the three species usually did not differ significantly in their susceptibility to either cypermethrin or to permethrin; however, with A. ipsilon, females were more susceptible to permethrin than to cypermethrin. Several instances of greater than additive toxicity were noted when insects were treated with piperonyl butoxide, S,S,S-tri-n-butyl phosphorotrithioate (DEF), or amitraz 30 min before cypermethrin. DEF exhibited the broadest spectrum of synergistic activity.     

喜树碱对草地贪夜蛾幼虫中肠细菌群落组成及多样性的影响

草地贪夜蛾Spodoptera frugiperda(J.E.Smith)是一种重要的入侵害虫,对多种作物生产造成严重的威胁.喜树碱是一种单萜类吲哚生物碱,对草地贪夜蛾幼虫生长发育具有显著抑制效果.本研究采用饲料混药法将喜树碱以1 mg/kg浓度处理草地贪夜蛾4龄幼虫3 d,解剖幼虫收集中肠内容物后采用16S rDNA...