Purification and properties of cytochrome b5 from the larval midgut of the southern armyworm (Spodoptera eridania)

The cytochrome b₅-like protein in the microsomal fraction of southern armyworm (Spodoptera eridania) larval midguts was solubilized with bromelain and purified about 130-fold to a specific content of 55.4 nmoles per mg protein. The protein had protoheme as its prosthetic group, a molecular weight of 11,400 ± 600 and its amino acid content and spectral properties were similar to those of cytochrome b₅ purified from rat liver microsomes by a similar procedure. The midpoint oxidation-reduction potential (E₀^′) was −57 mV. The results strongly suggest that the armyworm midgut cytochrome is a structurally similar protein to mammalian cytochrome b₅.     

Purification and characterization of NADPH--cytochrome c reductase from the midgut of the southern armyworm (Spodoptera eridania).

1. NADPH-cytochrome c reductase was solubilized with bromelain and purified about 400-fold from sucrose/pyrophosphate-washed microsomal fractions from southern armyworm (Spodoptera eridania) larval midguts. 2. The enzyme has a mol.wt. of 70 035 +/- 1300 and contained 2 mol of flavin/mol of enzyme consisting of almost equimolar amounts of FMN and FAD. 3. Aerobic titration of the enzyme with NADPH caused the formation of a stable half-reduced state at 0.5 mol of NADPH/mol of flavin. 4. Kinetic analysis showed that the reduction of cytochrome c proceeded by a Bi Bi Ping Pong mechanism. 5. Apparent Km values for NADPH and cytochrome c and Ki values for NADP+ and 2'-AMP were considerably higher for the insect reductase than for the mammalian liver enzyme. 6. These are discussed in relation to possible differences in the active sites of the enzymes.     

A cardioactive peptide from the southern armyworm, Spodoptera eridania

A cardioactive peptide was isolated from extracts of whole heads of the southern armyworm, Spodoptera eridania. This peptide has the sequence ENFAVGCTPGYQRTADGRCKPTF (Mr = 2516.8), determined from both Edman sequencing and tandem mass spectrometry in combination with off-line micropreparative capillary liquid chromatography. This peptide, termed Spoer-CAP23, has excitatory effects on a semi-isolated heart from larval Manduca sexta, causing an inotropic effect at low concentrations of peptide and chronotropic and inotropic effects at high doses. The threshold concentration for stimulatory effects of the synthetic peptide on the semi-isolated heart was about 1 nM, suggesting a physiological role as a neuropeptide.     

Aryl hydrocarbon hydroxylase in larvae of the southern armyworm (Spodoptera eridania)

Aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) activity in midgut microsomes of southern armyworm (Spodoptera eridania) larvae was induced 11-fold and 5.6-fold respectively following three days of feeding on diets containing pentamethylbenzene or naphthalene (both 0.2% w/v). β-Naphthoflavone and Aroclor 1254 were less effective inducers of AHH activity, phenobarbital was only slightly active and 3-methylcholanthrene caused a decrease in enzyme activity. AHH activity in microsomes from untreated and induced larvae was susceptible to inhibition by α-naphthoflavone, 1-phenylimidazole and piperonyl butoxide. Equilibrium dialysis studies with 1-(4′-³H-phenyl)imidazole showed that control and induced armyworm midgut microsomes contained a class of cytochrome(s) P-450 with a uniformly high affinity for phenylimidazole. It is concluded that AHH activity in the armyworm is catalyzed by a class of cytochrome(s) P-450 with characteristics intermediate between mammalian cytochrome(s) P-450 and P-448.     

Induction by carrot allelochemicals of insecticide-metabolising enzymes in the southern armyworm (Spodoptera eridania)

Carrot foliage monoterpenes induce cytochrome P-450 up to 2.9-fold, NADPH cytochrome c (P-450) reductase up to 1.6-fold, NADPH-oxidation up to 3.8-fold, aldrin epoxidation up to 1.5-fold in southern armyworm larval midgut tissues when incorporated in their diet at 0.2% for 3 days. Stigmasterol and ergosterol did not substantially induce microsomal oxidase activities and significantly inhibited GSH S-aryltransferase activity and sulfotransferase activity. Coumarin did not substantially affect microsomal oxidase and sulfotransferase activity but is the most potent inducer of GSH S-aryltransferase activity, increasing this activity 7-fold. None of the chemicals is acutely toxic to the sixth instar larvae or affect the larval weight gain except coumarin which significantly depressed the maximal body weight attained.     

警惕南方黏虫传入我国

原产于美洲热带的南方黏虫Spodoptera eridania(Stoll)是一种多食性的农业害虫。2016年南方黏虫入侵到非洲4个国家,对木薯和番茄等作物造成了严重危害,目前在我国尚未发现该害虫及相关报道。本文综述了南方黏虫的形态特征、地理分布、生物学特性以及防治措施,为我国今后南方黏虫的检测、监测和防治工作提供参考。从适生性度看,南方黏虫的寄主植物在我国广泛分布;我国也存在气候环境与该虫生存条件相似的地区,且寄主植物所在区域的气候条件同样适于南方黏虫的生长发育。伴随着国际贸易和其自身强大的迁飞能力,南方黏虫具有入侵我国的可能性。因此,有必要对其入侵我国的威胁提出预警,加强边境口岸检疫是防范该虫入侵的主要手段。     

Enzymic sulphation of p-nitrophenol and steroids by larval gut tissues of the southern armyworm (Prodenia eridania Cramer)

1. An enzyme system that catalyses the sulphation of p-nitrophenol, cholesterol, alpha-ecdysone, beta-sitosterol, dehydroepiandrosterone, oestrone and four other steroids of plant and insect origin was obtained from the soluble fraction of southern-armyworm gut tissues. 2. The enzyme system required ATP and inorganic sulphate, and activity was slightly enhanced in the presence of GSH. 3. The properties of this enzyme system with respect to pH, temperature, substrate and protein concentrations and various cofactors and reagents were studied. At -23 degrees C the enzyme preparation could be stored for 2 weeks without drastic loss of activity. At the end of storage for 1 month the loss of activity was approx. 21%. 4. The possible involvement of this enzyme system in insect endocrine control is discussed.     

An endogeneous inhibitor of microsomal mixed-function oxidases in homogenates of the southern armyworm (Prodenia eridania)

1. A potent inhibitor of microsomal mixed-function oxidation has been isolated from the gut contents of larvae of the southern armyworm (Prodenia eridania). 2. Progressive inhibition is associated with a soluble proteinase with a molecular weight of approx. 26000. 3. Although the proteolytic activity of the inhibitor can be reduced by phenylmethanesulphonyl fluoride and other reagents, these materials are only partially effective in protecting the microsomal enzymes. 4. Inhibitory activity is decreased in the presence of bovine serum albumin.     

Identification,accumulation, and biosynthesis of the cuticular hydrocarbons of the southern armyworm,Spodoptera eridania (cramer) (lepidoptera: Noctuidae)

The accumulation and biosynthesis of cuticular and internal hydrocarbons in the Southern armyworm, Spodoptera eridania, were examined at closely timed intervals during larval and pupal development. Gas chromatography-mass spectrometry (GC-MS) was used to identify n-alkanes, monomethylalkanes, and dimethylalkanes ranging in chain length from 23 to 35 carbons. The amount of cuticular hydrocarbon stayed relatively constant during each stadium, while the amount of internal hydrocarbon increased dramatically during the first half of each larval stadium, presumably to replace the cuticular hydrocarbon lost on the shed cast skin with each molt. The accumulation of internal hydrocarbon was mirrored by large increases in the rate of incorporation of labeled acetate into the hydrocarbon fraction. Hydrocarbon production fell to very low rates during the latter part of the fourth and fifth larval stadia. Relatively high rates of hydrocarbon production were observed during the first and last one-third of the pupal stage and essentially all of the hydrocarbons produced during this stage remained internal. These data document large changes in the rates of hydrocarbon production during development in S. eridania and suggest that most of the hydrocarbon produced during each stage was stored internally and then transported to the cuticle of the next stage.     

Properties of 5-aminolaevulinate synthetase and its relationship to microsomal mixed-function oxidation in the southern armyworm (Spodoptera eridania).

1. Activity of 5-aminolaevulinate synthetase was measured in the midgut and other tissues of the last larval instar of the southern armyworm (Spodoptera eridania Cramer, formerly Prodenia eridania Cramer). 2. Optimum conditions for measuring the activity were established with respect to all variables involved and considerable differences from those reported for mammalian enzyme preparations were found. 3. Maximum activity (20 nmol/h per mg of protein) occurs 18-24 h after the fifth moult and thereafter decreases to trace amounts as the larvae age and approach pupation. 4. Synthetase activity was rapidly induced by oral administration (in the diet) of pentamethylbenzene, phenobarbital, diethyl 1,4-dihydro-2,4,6-trimethylpyridine-3, 5-dicarboxylate, and 2-allyl-2-isopropylacetamide. 5. Puromycin inhibited the induction of synthetase by pentamethylbenzene. 6. Induction of 5-aminolaevulinate synthetase correlated well with the induction of microsomal N-demethylation of p-chloro-N-methylaniline, except for phenobarbital, which induced the microsomal oxidase relatively more than the synthetase.     

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.     

Association of a fungal endophyte in perennial ryegrass with antibiosis to larvae of the southern armyworm, Spodoptera eridania

Larvae of the Southern armyworm Spodoptera eridania Cramer (Lepidoptera:Noctuidae), feeding on perennial ryegrasses (Lolium perenne L.) infected with an endophytic fungus (Acremonium loliae Latch, Christensen and Samuels), had a much lower survival rate (7–13%) than larvae feeding on endophyte-free ryegrasses (82–90%). Death of the larvae on endophyte-infected entries occurred rapidly between 144 h and 168 h of feeding. This corresponded with armyworms feeding on the base of the plant, where endophyte concentration is highest. Twenty-four hours after the start of the bioassay the larval mass and rate of larval development were significantly higher on endophytic entries. From 48–144 h no differences were seen, but after 144 h the mass of larvae on endophyte-infected grasses sharply declined. Observations from this bioassay further substantiate the association between A. loliae-infected ryegrass and antibiosis to several lepidopterous and coleopterous insect pests.

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Résumé Le ray-grass vivace (Lolium perenne), contaminé par le champignon endophyte Acremonium loliae Latch, Christensen & Samuels, a présenté une augmentation de la resistance à de nombreux coléoptères et lépidoptères nuisibles. Cette note examine les réactions de Spodoptera eridania Cramer (Lépido., Noctuidae) alimenté sur trois lignées de ray-grass contaminées par le champignon et trois lignées saines. Après 168 h d'alimentation sur ray-grass contaminé, les chenilles presentent une très forte mortalité; la survie n'est que de 7 à 13% contre 82 à 90% pour le ray-grass sain. Le décès brutal des chenilles correspond à leur alimentation sur la base de la plante ou la concentration du champignon est la plus forte. Les chenilles consomment constamment, broutant plus des 2/3 du feuillage du ray-grass; les broutements des six séries ne différaient pas significativement.Au bout de 24 h, la nombre de chenilles passées du 3ème au 4ème stade, et l'augmentation de poids sont plus élevés pour les séries sur plante contaminée, ce qui suggère un avantage initial pour les chenilles en présence de champignon endophyte, l'analyse en poids sec a montré que l'augmentation de poids initial est réel. Entre 48 et 144 h, cependant, le nombre de 4ème stade et le poids des chenilles sont les mêmes dans les deux séries. Après 144 h, le poids des chenilles sur ray-grass contaminé diminue significativement; aucune n'était parvenue au 5ème stade, contre 11% sur ray-grass sain. Nous n'avons pas observé de signes apparents de neurotoxicité. Au lieu de cela, il ya a eu interaction avec un processus physiologique fondamental, ce qui a provoqué une forte perte de poids larvaire et la mort, indiquant l'intervention d'inhibiteurs métaboliques.

     

Antibiosis/antixenosis in tulip tree and quaking aspen leaves against the polyphagous southern armyworm,Spodoptera eridania

Summary Previous studies have shown leaves of tulip tree, Liriodendron tulipifera L. (of the Magnoliaceae) and of Populus tremuloides Michx. (of the Salicaceae) to be antixenotic/antibiotic to many Lepidoptera, including one of the most polyphagous of all phytophagous insects, the southern armyworm, Spodoptera eridania Cramer (Noctuidae). We investigated the physiological responses to this phytochemical activity on neonate and late instar armyworm larvae in controlled environments with particular emphasis upon the leaf extracts containing condensed tannins and hydrolysable tannins. These tannin-containing extracts of tulip tree leaves and quaking aspen leaves were generally toxic to neonate larvae. For later instars, growth suppression was not due to digestibility-reduction, but instead to suppressed consumption rates and greatly increased metabolic (respiratory) costs as reflected in reduced biomass conversion efficiencies.     

Cyanide as a feeding stimulant for the southern army worm, Spodoptera eridania

ABSTRACT.

• 1 All instars of Spodoptera eridania larvae grow as well or better when cyanide is present in their diet as when it is absent. Concentrations up to 0.05% stimulate feeding in first to fourth instar larvae. Concentrations from 0.1% to 1.0% stimulate feeding in fifth and sixth instar larvae.
• 2 Three-day-old sixth instar larvae pre-exposed to cyanide are completely resistant to its acutely toxic effects, but previously unexposed larvae suffer reversible symptoms of poisoning when feeding on a diet containing 1.0% KCN.
• 3 A 1.0% dietary KCN exposure during the sixth instar reduces ecdysis to 17% adult emergence and completely inhibits oviposition.
• 4 Cyanide concentrations from 0.5% to 1.0% in the diet, although effecting increased growth rates, induce necrotic lesions in larval mid-gut epithelial cells.
• 5 Thiocyanate, one of the in vivo cyanide metabolites, at 0.5% in the diet reduces pupation to 23%, delays and reduces adult emergence to 20% and inhibits oviposition.
• 6 The preferred host plant of S.eridania is the lima bean, Phaseolus lunatus, probably due to its content of the cyanogenic glycoside linamarin. Dietary valine has no effect on the southern armyworm feeding and growth behaviour (Long & Brattsten, 1982) but dietary cyanide does. The lima bean is known to contain up to 31 ppm cyanide in some varieties.

     

Mechanism of inhibition of microsomal mixed-function oxidation by the gut-contents inhibitor of the southern armyworm (Prodenia eridania)

A potent inhibitor of microsomal mixed-function oxidation reactions in insects had previously been isolated and partially purified from the gut contents of Prodenia eridania and shown to be associated with proteinase activity. Incubation of rat liver microsomal fraction with low concentrations of this inhibitor led to solubilization of NADPH–cytochrome c reductase, which was paralleled by the inactivation of reduction of cytochrome P-450 by NADPH and by the inhibition of NADPH-linked benzo[3,4]pyrene hydroxylation and aminopyrine demethylation. There was little or no effect on cytochromes b₅ and P-450, nor was the capacity of the latter catalyst to combine with exogenous substrates decreased. Contrary to the findings with NADPH, preincubation of microsomal fraction with the inhibitor did not cause a significant decrease in the rate of cytochrome P-450 reduction by NADH, supporting the assumption that different catalysts are involved in the electron transfer from NADH and NADPH to cytochrome P-450. The findings indicate the importance of taking the possible presence of endogenous inhibitors into consideration when evaluating low or absent mixed-function oxidation activities found in insect systems in vitro.