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.     

Effects of feeding Spodoptera littoralis on lima bean leaves: IV. Diurnal and nocturnal damage differentially initiate plant volatile emission

Continuous mechanical damage initiates the rhythmic emission of volatiles in lima bean (Phaseolus lunatus) leaves; the emission resembles that induced by herbivore damage. The effect of diurnal versus nocturnal damage on the initiation of plant defense responses was investigated using MecWorm, a robotic device designed to reproduce tissue damage caused by herbivore attack. Lima bean leaves that were damaged by MecWorm during the photophase emitted maximal levels of beta-ocimene and (Z)-3-hexenyl acetate in the late photophase. Leaves damaged during the dark phase responded with the nocturnal emission of (Z)-3-hexenyl acetate, but with only low amounts of beta-ocimene; this emission was followed by an emission burst directly after the onset of light. In the presence of (13)CO(2), this light-dependent synthesis of beta-ocimene resulted in incorporation of 75% to 85% of (13)C, demonstrating that biosynthesis of beta-ocimene is almost exclusively fueled by the photosynthetic fixation of CO(2) along the plastidial 2-C-methyl-D-erythritol 4-P pathway. Jasmonic acid (JA) accumulated locally in direct response to the damage and led to immediate up-regulation of the P. lunatus beta-ocimene synthase gene (PlOS) independent of the phase, that is, light or dark. Nocturnal damage caused significantly higher concentrations of JA (approximately 2-3 times) along with enhanced expression levels of PlOS. Transgenic Arabidopsis thaliana transformed with PlOS promoter :: beta-glucuronidase fusion constructs confirmed expression of the enzyme at the wounded sites. In summary, damage-dependent JA levels directly control the expression level of PlOS, regardless of light or dark conditions, and photosynthesis is the major source for the early precursors of the 2-C-methyl-D-erythritol 4-P pathway.     

Effects of feeding Spodoptera littoralis on lima bean leaves. II. Continuous mechanical wounding resembling insect feeding is sufficient to elicit herbivory-related volatile emission

Herbivore feeding elicits defense responses in infested plants, including the emission of volatile organic compounds that can serve as indirect defense signals. Until now, the contribution of plant tissue wounding during the feeding process in the elicitation of defense responses has not been clear. For example, in lima bean (Phaseolus lunatus), the composition of the volatiles induced by both the insect caterpillar Spodoptera littoralis and the snail Cepaea hortensis is very similar. Thus, a mechanical caterpillar, MecWorm, has been designed and used in this study, which very closely resembles the herbivore-caused tissue damage in terms of similar physical appearance and long-lasting wounding period on defined leaf areas. This mode of treatment was sufficient to induce the emission of a volatile organic compound blend qualitatively similar to that as known from real herbivore feeding, although there were significant quantitative differences for a number of compounds. Moreover, both the duration and the area that has been mechanically damaged contribute to the induction of the whole volatile response. Based on those two parameters, time and area, which can replace each other to some extent, a damage level can be defined. That damage level exhibits a close linear relationship with the accumulation of fatty acid-derived volatiles and monoterpenes, while other terpenoid volatiles and methyl salicylate respond in a nonlinear manner. The results strongly suggest that the impact of mechanical wounding on the induction of defense responses during herbivore feeding was until now underestimated. Controlled and reproducible mechanical damage that strongly resembles the insect's feeding process represents a valuable tool for analyzing the role of the various signals involved in the induction of plant defense reactions against herbivory.     

Effect of zinc nutritional status on activities of superoxide radical and hydrogen peroxide scavenging enzymes in bean leaves

The effect of varied zinc (Zn) supply on the activities of superoxide dismutase (SOD), ascorbate (AsA) peroxidase, glutathione (GSSG) reductase, catalase and guaiacol peroxidase was studied in leaves of bean (Phaseolus vulgaris) plants grown for 15 days in nutrient solution. Zinc deficiency severely decreased plant growth and the leaf concentrations of soluble protein and chlorophyll. Resupply of Zn to deficient plants for up to 72h restored protein concentrations more rapidly than chlorophyll and plant growth. With the exception of guaiacol peroxidase, the activities of all enzymes were significantly decreased by Zn deficiency, in particular GSSG reductase and SOD. Within 72h of resupplying Zn to deficient plants, the enzyme activities reached the level of the Zn sufficient plants. The results indicate severe impairment in the ability of Zn-deficient leaves to enzymically scavenge O₂ ^- and H₂O₂. Consequences and reasons of this impairment are discussed in terms of photooxidation of chloroplast pigments and inhibition of the biosynthesis of the related scavenger enzyme proteins.     

Decreased membrane integrity in castor bean mitochondria by hydrogen peroxide. Evidence for the involvement of phospholipase D.

Purified mitochondria from germinating castor bean (Ricinus communis L.) endosperm was treated with hydrogen peroxide (H₂O₂), active oxygen form, in order to investigate the extent of membrane degradation. Incubation of mitochondria with micromolar concentrations (50–200 μM) of H₂O₂ resulted in a concentration-dependent loss of membrane proteins. During this process extensive loss of lipid-phosphate content was also observed in mitochondrial membranes. When L-3-phosphatidyl[2-¹⁴C]ethanolamine was added to the mitochondrial membranes as an exogenous substrate, the level of radioactivity in the water-soluble fraction was markedly enhanced with increasing concentration of H₂O₂. Analysis of the water-soluble products formed during the metabolism of ethanolamine-labelled phosphatidylethanolamine by mitochondrial membranes from castor bean indicates that this loss of lipid-phosphate is attributable to action of phospholipase D. Direct measurement of mitochondrial phospholipase D indicated that the activity of enzyme was remarkably stimulated by calcium ion or sodium dodecylsulfate (SDS). The optimum concentrations for enzyme stimulation were 25 and 0.5 mM for calcium ion and SDS in the reaction mixture, respectively. The substrate specificity of phospholipase D was determined by comparing various classes of exogenous phospholipids, added in the form of sonicated vesicles, as substrates. The phospholipase D exhibited preference for phosphatidylethanolamine. Taken together, our results suggest that increase of mitochondrial phospholipase D activity may be a key event leading to accelerated membrane deterioration following active oxygen attack.     

西花蓟马取食与机械损伤对菜豆叶片抗氧化系统的影响

本文研究了西花蓟马Frankliniella occidentalis(Pergande)取食和机械损伤诱导对菜豆抗氧化系统的影响,比较了不同损伤形式诱导的抗氧化酶活力和抗氧化物质含量的变化差异。结果表明,西花蓟马取食和机械损伤均可诱导菜豆叶片内过氧化物酶(POD)、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)3种抗氧化酶活性有不同程度的升高,但两种处理诱导的抗氧化酶活性变化规律不完全相同,各种酶活性达到最高点时间不同,西花蓟马取食对抗氧化酶活力的诱导作用大于机械损伤的。两种处理诱导下的类胡萝卜素的含量变化不大,但不论西花蓟马取食还是机械损伤均导致菜豆叶片内类黄酮和总酚含量整体呈下降趋势,西花蓟马的取食诱导的下降幅度大于机械损伤的。因此,西花蓟马取食诱导明显高于机械损伤对菜豆抗氧化系统的影响。     

Sequential diets,metabolic costs,and growth of Spodoptera eridania (Lepidoptera: Noctuidae) feeding upon dill,lima bean,and cabbage

Summary This study illustrates the diversity of feeding responses of individually polyphagous southern armyworms, Spodoptera eridania, to plants with differing allelochemics. In spite of the near optimal leaf water and nitrogen contents of the young foliage, it is apparent that vastly different larval growth performance results from dill, lima bean, and cabbage. Cabbage is the poorest food (as measured by larval growth rates and metabolic costs of processing the plant biomass). Unlike the case with certain other plant species or cultivars that are costly to process, with cabbage, S. eridania does not compensate for low efficiencies (E.C.D.'s) with increased consumption rates (R.C.R.'s). Biochemical or physiological reasons for this inability are unknown.A sequence of foods (changed each 18–24 h) apparently did not add sufficient stress upon the MFO system to be detected in the respiratory expenditures of S. eridania larvae, in spite of the fact that dill is known to contain insecticidal and synergistic chemicals (Lichtenstein et al. 1974). The larval growth performances and metabolic expenditures in these sequences were intermediate between the best food (dill) and the worse (cabbage). Significant differences were observed however between the sequential switching sequences, perhaps indicating that particular periods during the instar are especially more sensitive to certain allelochemics. Actual respiratory costs of the lima bean-cabbage-dill (i.e. B-C-D) sequence were 40–50% higher than observed for the other two sequences and more than 50% higher than the theoretical metabolic costs based on the proportions actually eaten and known costs associated with each food.This study and a related one (Scriber 1981a) illustrate how consumption rates, feeding efficiences, and larval growth of Spodoptera eridania are not species, population, or even individual characteristics, (cf. Fox and Morrow 1981), but instead depend largely upon variations in plant allelochemics and plant nutritional quality (Wolfson 1978; Scriber, 1981 b; Scriber and Slansky 1981). More significantly they illustrate that the food consumed in earlier instars (Scriber 1981 a) as well as the food consumed earlier in an instar can be a major influence upon the observed armyworm growth performances under a given set of environmental conditions.A publication of the College of Agriculture and Life Sciences, University of Wisconsin, Madison 53706Mention of a product or brand name does not reflect an endorsement of that item by the University of Wisconsin     

Generation of hydrogen peroxide by cerebral-cortex synaptosomes. Stimulation by ionomycin and plasma-membrane depolarization

Guinea-pig cerebral cortex synaptosomes steadily release H2O2 into the suspending medium, at the rate of 20-30 pmol min-1 mg protein-1. A transient increase of the H2O2 release is induced by the addition of 1 mM Ca2+, which declines within 60-90 s to a rate identical or slightly higher than that before Ca2+. The extra H2O2 following Ca2+ addition varies between 40-100 pmol/mg protein and is insensitive to verapamil. The H2O2 release increases strongly (up to 250 pmol min-1 mg-1) upon depletion of the synaptosomal glutathione by treatment with 1-chloro-2,4-dinitrobenzene, a substrate for glutathione transferase. This treatment however has no effect on the Ca2+-induced H2O2 transient. In these treated synaptosomes a further increase of the output of H2O2 is rapidly induced upon addition of the Ca2+ ionophore ionomycin. This increase (about 100 pmol min-1 mg-1) lasts several minutes and requires the presence of Ca2+. A similar, though less pronounced increased H2O2 release is obtained (also in the absence of Ca2+) upon depolarization of the synaptosomal plasma membrane with KCl or with veratridine.     

3''-phosphoadenosine-5''-phosphosulphate synthesis and involvement in sulphotransferase reactions in the insect, Spodoptera littoralis.

1. Synthesis of 3'-phosphoadenosine-5'-phosphosulphate from ATP and 35SO4(-2) was demonstrated by homogenates of gut. Malpighian tubules and fat body of Spodoptera littoralis. 2. The enzyme system was most active in the gut tissue, and was primarily located in the cytosol fraction of the cell. Gut cytosol preparations were used as a source of the 3'-phosphoadenosine-5'-phosphosulphate generating system for more detailed studies. 3. Maximum synthesis required an incubation mixture containing Tris/HCl buffer (pH 7.5), ATP (20 mM), MgCl2 (13.0 mM) and K2SO4 (3 mM). 4. The specific activity of 3'-phosphoadenosine-5'-phosphosulphate synthesizing activity in gut cytosol increased during development of the sixth instar larva, reaching a peak at day 4. A sudden fall in specific activity was observed in the prepupal stage. 5. 3'-Phosphoadenosine-5'-phosphosulphate formation is the rate limiting process in the overall sulphation of p-nitrophenol in the gut cytosol preparations from S. littoralis. 6. It is concluded that the properties of the sulphate-activating system in this insect are similar to those reported for vertebrates.     

Effects of Bt maize expressing Cry1Ab and Bt spray on Spodoptera littoralis

Various studies have been conducted to assess the damage caused by secondary lepidopteran pests to transgenic Bt maize expressing Cry1Ab. However, to date little is known on the effects of transgenic maize on Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae), a polyphagous herbivore which is considered a pest in Mediterranean maize growing areas. Here we present results on the effects of Bt maize (Bt‐11) and Bt spray (Dipel) on the various life stage parameters of this herbivore. We further assess the expression of Cry1Ab in different leaves and leaf parts in maize at a given plant growth stage, and determine whether the feeding damage of 3rd instar S. littoralis is influenced by Bt toxin expression. Contrary to previous literature reporting that S. littoralis is not sensitive to Bt Cry1Ab toxin, our results show that insects fed on either transgenic or Bt sprayed plants were negatively affected. Young S. littoralis larvae (1st and 2nd instars) were found to be the most sensitive to the Bt toxin. This was represented by a higher mortality and a slower developmental time of larvae maintained on transgenic or sprayed plants when compared to insects maintained on control plants. Moreover, Bt maize had a stronger and prolonged detrimental effect on insects when compared to Bt spray in maize. This was revealed by the fact that insects maintained on transgenic plants from 3rd instar to pupation took longer to reach adult emergence compared to insects that were maintained on sprayed plants. This was likely due to the continuous exposure of insects to the toxin when kept on transgenic maize. ELISA results showed a variation in the amount of Bt toxin among different leaf sections in transgenic maize at a given plant growth stage. These differences in Bt toxin were primarily found in the youngest leaf of growing plants. Although the lowest amounts of Bt toxin were detected in the growing leaf section of young leaves, this difference did not appear to influence the feeding behavior of 3rd instar S. littoralis.     

Contrasting consequences of plant domestication for the chemical defenses of leaves and seeds in lima bean plants

Plant domestication is assumed to result in reduced levels of defensive compounds in crops, because this makes the plants more suitable for consumption by humans and livestock. We argue that this should mainly be reflected in the concentrations of defense compounds in the plant parts that are used for consumption and not necessarily for other parts of crop plants. We tested this hypothesis for domesticated lima bean (Phaseolus lunatus), by comparing its chemical defenses against a leaf herbivore, the beet armyworm (Spodoptera exigua), and a seed predator, the beetle Zabrotes subfasciatus. For seeds and leaves we determined the concentrations of cyanogenic glycosides (CNGs) in cultivated varieties and wild populations and evaluated the preference and performance of the herbivores when exposed to leaves and seeds from wild and cultivated plants. Concentrations of CNGs were significantly different between wild and cultivated plants. In the leaves the concentration of CNGs in the cultivated varieties were more than double that of the wild leaves. In contrast, seeds from cultivated plants had up to 20 times lower CNG concentration compared to seeds from the wild populations. Insect preference and performance do not parallel the chemical data. Larvae of S. exigua preferred wild leaves but had higher survival on cultivated leaves. The beetles, however, strongly preferred seeds from cultivated plants and females developed more quickly on these seeds. We conclude that domestication of P. lunatus has altered the concentration of CNGs in both the seeds and the leaves in opposite directions. This results in differential effects on the herbivores that attack these two plant structures. The contrasting effect of domestication on different plant tissues can be explained by the fact that bean plants have been specifically selected for human consumption of the seeds. Tissue-specific effects of plant domestication on plant defenses can be expected for other crops as well.     

Valency-dependent stimulating effects of lima bean lectins on lymphocytes of different species.

Di- and tetravalent lectins purified from lima beans have mitogenic activity towards human, bovine, rabbit, rat and probably mouse lymphocytes; the effect of the mitogen varies for the different species. The mitogenic activity of the 2 lima bean lectins is related to their valency: LIM 124, the component with molecular weight 124 000 and 2 saccharide binding sites, is a weak mitogen; LIM 247, the component with molecular weight 247 000 and four saccharide binding sites, is several times more active. There are indications that the tetravalent LIM 247 exhibits B cell stimulatory activity.     

Significance of penetration and metabolism on topical toxicity of diflubenzuron in Spodoptera littoralis and Spodoptera exigua

Toxicity assays indicated that when diflubenzuron (DFB) was topically applied on larvae of the Egyptian cotton leafworm, Spodoptera littoralis (Boisduval), and the beet armyworm, Spodoptera exigua (Hübner), estimated LD₅₀ values were similar, reaching 0.47 and 0.44 ng/larva, respectively. This study evaluated the importance of the rate of uptake and excretion, and of enzymatic metabolism in building up an insecticidal toxicity after topical application on the insect cuticle in both species. In general, penetration of DFB in S. littoralis was about 2-fold higher than in S. exigua, whereas metabolic breakdown was of minor importance in S. littoralis as compared with S. exigua. The current data indicate the importance of cuticle penetration and discuss that extrapolation from oral feeding toxicity results to topical toxicity data should be evaluated with caution, especially for insect growth regulators used in integrated pest management.     

Effects of light and temperature on the circadian system controlling sperm release in moth Spodoptera littoralis

Reproductive physiology of male moths is regulated by a peripheral circadian system, which controls the timing of sperm release from the testis into the upper vas deferens (UVD) and timing of sperm transfer from the UVD to the seminal vesicles. We investigated various effects of light and temperature on sperm release and transfer rhythms in the moth Spodoptera littoralis. We report that both rhythms persist for up to 1 week in constant darkness without significant dampening and are also temperature compensated in the range from 20°C to 30°C. However, the duration of sperm retention in the UVD is temperature-dependent; consequently, temperature exerts a masking effect on the rhythm of sperm transfer. Experimental manipulations of light and temperature regime demonstrated that light dominates over temperature in entraining the timing of sperm release and transfer. Nevertheless, temperature plays a critical role in the absence of light Zeitgeber. Sperm release and transfer are arrhythmic in constant light (LL); however, both rhythms are restored by temperature cycles.     

Cardenolides from Gomphocarpus sinaicus and Pergularia tomentosa (Apocynaceae: Asclepiadoideae) deter the feeding of Spodoptera littoralis

Methanol extracts of Gomphocarpus sinaicus, Pergularia tomentosa and Cynanchum acutum (Apocynaceae, sub-family Asclepiadoideae) deterred feeding of Spodoptera littoralis in a binary-choice bioassay. Analyses of extracts using high-performance liquid chromatography with photodiode array detection indicated that methanol extracts of P. tomentosa and G. sinaicus contained cardenolides, while these compounds were not detected in extracts of C. acutum. Activity-guided fractionation of the methanol extracts of G. sinaicus and P. tomentosa resulted in the isolation of six cardenolides: 7,8-dehydrocalotropin, calotropin and coroglaucigenin 3-(6-deoxy-β-allopyranoside)-19-acetate (frugoside 19-acetate) from G. sinaicus, and coroglaucigenin, 16α-acetoxycalotropin and calactin from P. tomentosa. The isolation of 16α-acetoxycalotropin was a new report from P. tomentosa. Each of the 6 cardenolides deterred feeding by S. littoralis, while two cardenolide standards, digoxin and digitoxin, did not affect feeding. Differences among cardenolides in their effect on feeding were associated with specific structural features. C. acutum is the only one of the three species tested that is known to support the development of S. littoralis, although the development of larvae was delayed. The observed feeding deterrent activity of the cardenolide-free methanol extract of C. acutum would suggest that compounds other than cardenolides are responsible for the deterrent activity. These compounds, although deterrent in a short-term feeding assay, might not prevent long term feeding, thus allowing the larvae to develop on the plant.     

Effects on oviposition behaviour and larval development of Spodoptera littoralis by herbivore-induced changes in cotton plants

Herbivore feeding induces chemical defence responses in plants. In this study we investigate how herbivore-induced changes in cotton plants, Gossypium hirsutum L., influence the oviposition behaviour and larval development of Spodoptera littoralis Boisd. (Lepidoptera: Noctuidae). In two-choice experiments female moths preferred to oviposit on small plants (3–4 leaves) that had been fed on by 3rd to 4th instar larvae (72%) over non-damaged control plants. However, when using larger plants (8 to 10 true leaves) the preference was reversed, with only 31% of the eggs deposited on the induced plants. The same trend was found with plants that had been given a similar level of damage by 6th instar larvae. However, the difference between the treatments was in both cases smaller with only 60% of the eggs deposited on the preferred plant treatment. If cotton plants subjected to artificial damage were compared with undamaged plants, none of the treatments were preferred for oviposition. No significant difference was found in larval weight, pupal weight, survival, or development time, between larvae reared their entire development on leaves from induced or from non-induced plants.     

Plant-odour-specific receptor neurones on the antennae of female and male Spodoptera littoralis

Abstract. Receptor neurones with high selectivity and sensitivity to plant odours were found within short sensilla trichodea on the antenna of both female and male Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) by using single-sensillum recording techniques. In 112 sensilla from females and forty-one from males, twenty-four different receptor neurone types were characterized according to their specificity. Altogether, twenty-six plant and three sex pheromone compounds were tested. Receptor neurones responding with high specificity to flower odours, green leaf volatiles, oviposition deterrents and other general host plant odours were identified. In twenty-one receptor neurone types, responses were elicited by one or several plant compounds, and in three types responses were elicited by sex pheromone compounds. The majority of the receptor neurones responded to only one or two of the tested compounds. In general, only one of the two receptor neurones in a sensillum responded to any of the compounds tested. An exception was a receptor neurone responding to plant odours (green leaf volatiles) and another receptor neurone responding to a sex pheromone compound ([Z]-7-dodecenyl (acetate), which occurred in the same sensillum. The majority of the receptor neurones displayed a high sensitivity to plant odours. No morphological difference was identified the different sensillum types.     

Effects of pressure and temperature on the reactions of horseradish peroxidase with hydrogen cyanide and hydrogen peroxide.

Reactions of ferric horseradish peroxidase with hydrogen cyanide and hydrogen peroxide were studied as a function of pressure. Activation volumes are small and differ in sign (delta V = 1.7 +/- 0.5 ml/mol for peroxidase + HCN and -1.5 +/- 0.5 ml/mol for peroxidase + H2O2). The temperature dependence of cyanide binding to horseradish peroxidase was also determined. A comparison is made of relevant parameters for cyanide binding and compound I formation.