GABA, β-Alanine and Glycine in the Digestive Juice of Privet-Specialist Insects: Convergent Adaptive Traits Against Plant Iridoids

The privet tree, Ligustrum obtusifolium (Oleaceae), defends its leaves against insects with a strong lysine-decreasing activity that make proteins non-nutritive. This is caused by oleuropein, an iridoid glycoside. We previously found that some privet-specialist caterpillars adapt by secreting glycine in the digestive juice as a neutralizer that prevents the loss of lysine. Here, we extended the survey into 42 lepidopteran and hymenopteran species. The average concentration of glycine in digestive juice for 11 privet-feeding species (40.396 mM) was higher than that for 32 non-privet-feeding species (2.198 mM). The glycine concentrations exceeded 10 mM in 7 out of 11 privet-feeding species. In Macrophya timida (Hymenoptera), it reached 164.8 mM. Three out of the four remaining privet-feeding species had other amino acids instead. Larvae of a privet-specialist butterfly, Artopoetes pryeri (Lycaenidae), had a high concentration (60.812 mM) of GABA. In two other specialists, β-alanine was found. GABA, β-alanine, and glycine as well as alanine, amines, and ammonium ion inhibited the lysine decrease, indicating that amino residues are responsible for the inhibition. However, the three amino acids found in the specialists were far more effective (20 mM showed 80% inhibition) than the rest (>140 mM was required for 80% inhibition). Our results show a clear and rare case of the apparent convergent evolution of herbivores’ molecular adaptations of feeding on a plant with a chemical defense in a manner that minimizes the cost of adaptation. The novel role of GABA in plant-herbivore interactions shown here is probably the first reported non-neuronal role of animal-derived GABA.     

Responses of maxillary sensilla styloconica inBombyx mori to glucosides fromOsmunda japonica,a nonhost plant

We isolated glucosides from the royal fern,Osmunda japonica, which elicit a deterrent response in larvae ofBombyx mori. These compounds were absent in taro (Colocasia antiquorum) and castor-oil plant (Ricinus communis) leaves and did not evoke responses of sensory cells in the lateral and medial sensilla styloconica ofSpodoptera litura. This glucoside extract of the royal fern leaves stimulates receptors generally associated with deterrent. It is also possible that this compound may act as a behavioral deterrent, and from actual feeding tests, it is suggested that this compound may prevent feeding in some monophagous insects, such asBombyx mori. The deterrent glucoside possesses a noncyclic aglycon.     

Light-induced variation in phenolic levels in foliage of rain-forest plants

Levels of phenolic secondary metabolites in the leaves of four west African rain-forest plants,Acacia pennata, Cynometra leonensis, Diopyros thomasii, andTrema guineensis, were correlated with incident light intensity at both the inter- and intraindividual level. Enhanced phenolic levels under high light intensity appeared to be due to production of both polyphenolics (condensed and hydrolyzable tannins) and simple phenolics. InTrema guineensis, where it is possible to separate leaves in terms of both their age and the light incident upon them, condensed tannin production progressed differently during the development of sun and shade leaves, suggesting continuing production of new oligomers in the former but not in the latter. The results of this study suggest that the production of phenolics in relation to variation in incident light is a finely tuned process, which must be explained in terms of plant physiology and intermediate metabolism rather than in terms of resource allocation or a direct response to herbivory.     

Olea europaea chemicals repellent toDacus oleae females

The egg dispersion strategy of the olive fruit flyDacus oleae, which is dependent on chemicals from the fruit, was investigated. In particular, the exact role ofo-diphenolic compounds, such as the typical olive glucosides, oleuropein and demethyloleuropein, and their derivatives was clarified. It appears that the strong chemotactile repulsive effect exerted by the water fraction of crushed olives is due mainly to (E)-2-hexenal. Several compounds, such as-3,4-dihydroxyphenylethanol and other oleuropein derivatives, which exert a strong chemotactile repulsion, were newly identified or confirmed either in fresh olive juice or in olive mill waste water. This result confirms that the small droplets of olive juice, regurgitated just after egg laying by theD. oleae female all around the oviposition hole, actually prevent other females from ovipositing on the same fruit.     

Biological activity of the shrubBoscia senegalensis (PERS.) LAM. ex Poir. (Capparaceae) on stored grain insects

Biological activity of leaves, fruits and extract of the African shrubBoscia senegalensis (PERS.) LAM. ex Poir. was evaluated against five stored-grain insects. When added to cowpeas at 2–4% (w/w), fresh ground fruits and leaves caused 80–100% mortality inCallosobruchus maculants (F.) adults and significantly reduced both emergence and damage of the F₁ progeny. Acetone fruit extract exhibited a potent fumigant effect onProstephanus truncatus Horn, C.maculatus, andSitotroga cerealella OLIV.; with LT₅₀ values of 3.8, 2.3, and below 1.5 hr, respectively. LC₅₀ determination forB. senegalensis fruits and leaves as well as pure methylisothiocyanate (MITC) onTribolium castaneum HERBST,Sitophilus zeamais MOTSCH. andC. maculatus showed a differential response of the insects to plant parts or MITC. Quantitative dosage ofBoscia active components and LC₅₀ values obtained for the plant tissues, compared to those of pure molecules, indicate that the biological activity ofB. senegalensis is due to the liberation of MITC from a glucosinolate precursor glucocapparin contained inBoscia fruits and leaves.     

Effects of birch phenolics on a grazing and a browsing mammal: A comparison of hares

Mountain hares (Lepus timidus L.) commonly have high proportions of birch (Betula spp.) in their winter diets, whereas European hares (Lepus europaeus) do not. The effects of a birch extract added to laboratory diets offered to mountain hares and European hares on the digestibility and sodium balance were measured. The extract added contained total phenolics equivalent in amount to that occurring in diets containing 0, 40, 60 and 80% dry matter of birch twigs. Increasing dietary phenolic concentration led to reduced voluntary food intake and apparent protein digestibility in both hare species. No effects on apparent dry matter digestibility were observed. The highest concentration of birch extract caused severe sodium losses via the urine by European hares but not by mountain hares. The effects of the birch extract within the digestive system appear to be similar in the two hare species, whereas the mountain hare appears to be better adapted to the toxic effects that disrupted sodium balance in the European hare. These preliminary results suggest that detoxification rather than digestive abilityper se may contribute to different mammalian herbivores' feeding strategies.     

Study on Oleuropein Extraction from Olive Tree (Olea europaea) Leaves by means of SFE: Comparison of Water and Ethanol as Co-Solvent

The present study focuses on developing methods for olive leaf extraction and deals with obtaining extract, rich in oleuropein, which is the most abundant phenolic compound in olive leaves. Supercritical fluid extraction (SFE) was applied to the dried and ground olive leaves by using CO₂ as supercritical (SC) fluid in the presence of water and ethanol as co-solvent. The influences of operating parameters by means of co-solvent content (0-1 mL/min), temperature (50 and 100°C) and pressure (100-300 bar) on both extract and oleuropein yields were investigated. Quantitative analysis was performed by using a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) technique. The experimental results obtained by using SC-CO₂ alone were not satisfactory, and it was seen that addition of a polar modifier is necessary in order to improve yield and selectivity of the process. It was observed that CO₂ modified by water and ethanol showed nearly the same extract performance, where CO₂ modified by water is better for high oleuropein yield.     

Influence of Induced Plant Defenses in Cotton and Tomato on the Efficacy of Baculoviruses on Noctuid Larvae

Constitutive phenolase activity of plants has a profound ability to modulate disease in insects caused by baculoviruses. We investigated the influence of damage-induced plant phenolic oxidases in cotton and tomato on mortality caused by two different baculoviruses in their respective hosts, Heliothis virescens (L.) and Helicoverpa zea (Boddie). For both plant species, peroxidase (POD) and phenolic levels were predictive of larval mortality caused by baculoviruses. The higher the POD activity, the lower the mortality in both hosts. Different classes of phenolics (e.g., monohydroxyphenolics vs. catecholic phenolics) in combination with POD activity had different effects on the severity of viral disease depending upon mixture, which implies that viral efficacy is predictable only if total chemical content of the plants is specified. Inhibition of baculoviral disease by plant phenolase activity has potential implications for the compatibility of baculoviruses with induced resistance in IPM programs.     

Defensive Role of Tomato Polyphenol Oxidases against Cotton Bollworm (<Emphasis Type="Italic">Helicoverpa armigera</Emphasis>) and Beet Armyworm (<Emphasis Type="Italic">Spodoptera exigua</Emphasis>)

Tomato (Solanum lycopersicum) polyphenol oxidases (PPOs), enzymes that oxidize phenolics to quinones, have been implicated in plant resistance to insects. The role of PPO in resistance to cotton bollworm [Helicoverpa armigera (Hübner)] and beet armyworm [Spodoptera exigua (Hübner)] (Lepidoptera: Noctuidae) was evaluated. Consumption, weight gains, and mortality of larvae feeding on foliage of transgenic tomato lines overexpressing PPO (OP lines) and of larvae feeding on foliage of transgenic tomato lines with suppressed PPO (SP lines) were compared with consumption, weight gains, and mortality of larvae feeding on non-transformed (NT) plants. Increases in foliage consumption and weight gains were observed for cotton bollworms feeding on leaves of SP plants compared to NT and OP plants. PPO activity was negatively correlated with both weight gains and foliar consumption of cotton bollworm, substantiating the defensive role of PPO against this insect. Similarly, beet armyworm consumed less foliage (both young and old leaves) from OP plants than SP plants. Larvae feeding on OP leaves generally exhibited lower weight gains than those feeding on SP leaves. These results indicate that tomato PPO plays a role in resistance to both cotton bollworm and beet armyworm.     

Allelopathic potential ofnuphar lutea (L.) Sibth. & Sm. (Nymphaeaceae)

Aqueous extracts ofNuphar lutea (L.) Sibth. & Sm. leaves (blades plus petioles) and roots plus rhizomes were tested for allelopathic activity using lettuce seedling andLemna minor L. assay systems. The 12.5, 25, 125, and 250 parts per thousand (ppt) treatments of both extracts killed the lettuce seedlings. At 2.5 ppt of extract, radicle growth of lettuce was 29% of the control for leaves and 31% of the control for roots plus rhizomes.Lemna minor frond number was reduced to 34% of the control by the 25 ppt leaf extract and to 43% of the control by the 25 ppt roots plus rhizomes extract.L. minor was killed by concentrations of 125 ppt and above of both plant part extracts. As expected, the frond number and total chlorophyll content measured by theL. minor assay were highly correlated. Osmotic potentials below 143 mOsmol/kg had no influence onL. minor growth. Neither the osmotic potential nor the pH of the undiluted extracts ofN. lutea were in the range known to influence the growth of either lettuce seedlings orL. minor. Nuphar lutea extracts were many times more inhibitory than 16 other hydrophytes we previously examined.     

Activation of plant foliar oxidases by insect feeding reduces nutritive quality of foliage for noctuid herbivores

The foliage and fruit of the tomato plantLycopersicon esculentum contains polyphenol oxidases (PPO) and peroxidases (POD) that are compartmentally separated from orthodihydroxyphenolic substrates in situ. However, when leaf tissue is damaged by insect feeding, the enzyme and phenolic substrates come in contact, resulting in the rapid oxidation of phenolics to orthoquinones. When the tomato fruitwormHeliothis zea or the beet army-wormSpodoptera exigua feed on tomato foliage, a substantial amount of the ingested chlorogenic acid is oxidized to chlorogenoquinone by PPO in the insect gut. Additionally, the digestive enzymes of the fruitworm have the potential to further activate foliar oxidase activity in the gut. Chlorogenoquinone is a highly reactive electrophilic molecule that readily binds cova-lently to nucleophilic groups of amino acids and proteins. In particular, the —SH and —NH₂ groups of amino acids are susceptible to binding or alkylation. In experiments with tomato foliage, the relative growth rate of the fruitworm was negatively correlated with PPO activity. As the tomato plant matures, foliar PPO activity may increase nearly 10-fold while the growth rate of the fruitworm is severely depressed. In tomato fruit, the levels of PPO are highest in small immature fruit but are essentially negligible in mature fruit. The growth rate of larvae on fruit was also negatively correlated with PPO activity, with the fastest larval growth rate occurring when larvae fed on mature fruit. The reduction in larval growth is proposed to result from the alkylation of amino acids/protein byo-quinones, and the subsequent reduction in the nutritive quality of foliage. This alkylation reduces the digestibility of dietary protein and the bioavailability of amino acids. We believe that this mechanism of digestibility reduction may be extrapolatable to other plant-insect systems because of the ubiquitous cooccurrence of PPO and phenolic substrates among vascular plant species.     

Reassessment of the role of gut alkalinity and detergency in insect herbivory

Previously it was reported that significant amounts of the tomato phenolic, chlorogenic acid, were oxidized in the digestive system of generalist feedersSpodoplera exigua andHelicoverpa zea. The covalent binding of the oxidized phenolic (i.e., quinone) to dietary protein exerts a strong antinutritive effect against larvae. In this study, we examined the fate of ingested chlorogenic acid in larvalManduca sexta, a leaf-feeding specialist of solanaceous plants. Significant amounts of chlorogenic acid were bound to excreted protein byM. sexta when larvae fed on tomato foliage. However, in the case ofM. sexta we suggest that the strong alkalinity and detergency of the midgut may minimize the antinutritive effects of oxidized phenolics. The solubility of tomato leaf protein is significantly greater at pH 9.7, representative of the midgut ofM. sexta, than at pH 8.0, representative of the midguts ofH. zea and S. exigua. We suggest that this increase in solubility would compensate for any loss in bioavailability of essential amino acids caused by the covalent binding of chlorogenic acid to amino acids. Furthermore, lysolecithin, a surfactant likely to contribute to the detergent properties of the midgut fluid, was shown to enhance protein solubility as well as inhibit polyphenol oxidase activity. The adaptive significance of gut alkalinity and detergency is discussed.Approved by the Director of the Arkansas Agricultural Experiment Station.     

Plant Phenolics as Dietary Antioxidants for Herbivorous Insects: A Test with Genetically Modified Tobacco

High foliar phenolics are generally assumed to increase resistance to insect herbivores, but recent studies show that tobacco lines modified to over– and underexpress phenolics do not exhibit higher constitutive resistance to caterpillars. This is contrary to the expectation that ingestion of tobacco phenolics, particularly chlorogenic acid, should cause oxidative stress in herbivores. We investigated free radical production and antioxidant capacity of fresh crushed leaves of tobacco lines exhibiting over a sixfold difference in chlorogenic acid content to test whether high phenolic concentrations are associated with increased production of reactive oxygen species (ROS). The effects of in planta phenolic levels on feeding behavior, growth, biochemical markers of oxidative stress, and the antioxidant capacity of midgut fluid and hemolymph were assessed in tobacco budworm, Heliothis virescens. The experiments showed that high phenolic foliage was more prooxidant than low phenolic foliage, but the net balance in crushed tissue was antioxidant in comparison to buffer and the commercial antioxidant standard, Trolox. In H. virescens, the antioxidant capacity of midgut fluid was also powerful, and caterpillars fed high phenolic foliage did not exhibit the expected markers of oxidative stress in midgut tissues (altered ascorbate ratios, disulfides, or total hydroperoxides). Instead, hemolymph of larvae fed high phenolic foliage exhibited improved total Trolox equivalent antioxidant capacity (TEAC). These results suggest that the elevated foliar phenolics in some plants may have beneficial antioxidant properties for herbivorous insects, much as dietary phenolics do in mammals.     

Environmental and biotic factors affecting the phenolic content of different cultivars ofSorghum bicolor

Levels of phenolic acids in healthy plants ofSorghum bicolor differ considerably with cultivar and always decrease as the plant matures. Laboratory- and field-grown plants show significant differences in phenolics. Environmental factors, particularly light intensity, influence the concentration of phenolics in sorghum. Attack by insects and pathogenic fungi also increase the phenolic content of the plants to varying degrees dependent on the cultivar and the stage of growth of the plant.     

Phenolics in black oak bark and leaves

Catechin, quercitrin, robinin, quercetin 3-methyl ether, scopoletin, cholorogenic acid, several leucoanthocyanins, and condensed and hydrolyzable tannins were identified in bark and leaves ofQuercus velutina Lamarck. The concentrations of most phenolics in leaves increased as the growing season progressed, whereas those of most phenolics in bark remained essentially unchanged. Qualitative differences in bark and leaf phenolics among different trees were negligible.     

Tannin Composition Affects the Oxidative Activities of Tree Leaves

We examined whether tannin composition plays an important role in explaining the oxidative activities of tree leaves of Acer saccharum (sugar maple) and Quercus rubra (red oak). Sugar maple leaves contained substantial amounts of ellagitannins, condensed tannins, and galloyl glucoses, whereas red oak leaves contained almost exclusively condensed tannins. Oxidative activities of the crude phenolics from both species, and the phenolic fractions from sugar maple, were measured with electron paramagnetic resonance (EPR) spectrometry and UV-visible spectrophotometry. The two assays produced similar results: (1) sugar maple phenolics produced larger semiquinone radical concentrations,and higher semiquinone decay rates and browning rates than did red oak phenolics;(2) ellagitannin levels were positively associated with the three measures of oxidative activity; and (3) condensed tannin and galloyl glucose levels were negatively associated with these measures. The negative relationship between condensed tannin levels and oxidative activity resulted from the antioxidant effects of condensed tannins on hydrolyzable tannins; several purified condensed tannins significantly decreased the concentrations of semiquinone radicals and browning rates of pedunculagin (an ellagitannin) and pentagalloyl glucose. As expected, whole-leaf extracts from sugar maple produced elevated levels of semiquinone radicals, but none were observed in red oak extracts when the two species were compared with an EPR time-course assay. We conclude that the oxidative activities of tree leaves may be affected by tannin composition, and that the prooxidant activity of ellagitannins may be decreased by co-occurring condensed tannins.     

Phenolic Compounds in Red Oak and Sugar Maple Leaves Have Prooxidant Activities in the Midgut Fluids of <Emphasis Type="Italic">Malacosoma disstria</Emphasis> and <Emphasis Type="Italic">Orgyia leucostigma</Emphasis> Caterpillars

Phenolic compounds are generally believed to be key components of the oxidative defenses of plants against pathogens and herbivores. However, phenolic oxidation in the gut fluids of insect herbivores has rarely been demonstrated, and some phenolics could act as antioxidants rather than prooxidants. We compared the overall activities of the phenolic compounds in red oak (Quercus rubra) and sugar maple (Acer saccharum) leaves in the midgut fluids of two caterpillar species, Malacosoma disstria (phenolic-sensitive) and Orgyia leucostigma (phenolic-tolerant). Three hypotheses were examined: (1) ingested sugar maple leaves produce higher levels of semiquinone radicals (from phenolic oxidation) in caterpillar midgut fluids than do red oak leaves; (2) O. leucostigma maintains lower levels of phenolic oxidation in its midgut fluids than does M. disstria; and (3) phenolic compounds in tree leaves have overall prooxidant activities in the midgut fluids of caterpillars. Sugar maple leaves had significantly lower ascorbate:phenolic ratios than did red oak leaves, suggesting that phenolics in maple would oxidize more readily than those in oak. As expected, semiquinone radicals were at higher steady-state levels in the midgut fluids of both caterpillar species when they fed on sugar maple than on red oak, consistent with the first hypothesis. Higher semiquinone radical levels were also found in M. disstria than in O. leucostigma, consistent with the second hypothesis. Finally, semiquinone radical formation was positively associated with two markers of oxidation (protein carbonyls and total peroxides). These results suggest that the complex mixtures of phenolics in red oak and sugar maple leaves have overall prooxidant activities in the midgut fluids of M. disstria and O. leucostigma caterpillars. We conclude that the oxidative defenses of trees vary substantially between species, with those in sugar maple leaves being especially active, even in phenolic-tolerant herbivore species.     

Host-Plant Chemistry Influences Oviposition Choice of the Spicebush Swallowtail Butterfly

The spicebush swallowtail, Papilio troilus (L.), lays its eggs on plants in the family Lauraceae. Sassafras [Sassafras albidum (Nutt.) Nees], spicebush [Lindera benzoin (L.) Spreng.], redbay (Persea borbonia (L.)] and camphortree [Cinnamomum camphora (Nees) Eberm.] are four of its known host plants. In one-choice tests, free-flying spicebush swallowtail females laid eggs on chemical extracts of the leaves of each of these four hosts. In two-choice experiments, females always preferred to oviposit on an extract of sassafras compared to extracts of the other three hosts. It was shown for spicebush extract that this response was not due to oviposition experience. Previously we had identified one of the host plant chemicals acting as an oviposition stimulant in sassafras extract as 3-caffeoyl-muco-quinic acid (3-CmQA). Extracts of the other three hosts did not contain this compound. The addition of 3-CmQA alone to spicebush extract did not increase oviposition activity. It did, however, increase discrimination between hosts and nonhosts. When a fraction of sassafras extract containing 3-CmQA and other synergistic stimulants was added to spicebush extract, preference for sassafras extract was no longer recorded. These results show existing differences in oviposition chemistry among host plants of the spicebush swallowtail and how these differences can influence oviposition choice in bioassay experiments.     

Fate of the Dietary Terpene, P-Cymene, in the Male Koala

The fate of the dietary terpene, p-cymene, is reported in a specialist Eucalyptus leaf folivore, the koala (Phascolarctos cinereus). Six male koalas were given two single oral doses of p-cymene (0.37 mmol/kg and 1.49 mmol/kg). Urine and feces were collected for two 24-hr periods after each dose and analyzed for metabolites by extraction, gas chromatography, and mass spectrometry. A total of six metabolites were detected in the urine. A novel, extensively oxidized metabolite, 4-(1,2-dihydroxy-1-methylethyl)-benzoic acid, was identified and its structure elucidated by high resolution and chemical ionization mass spectrometry. Minor amounts of two glycine conjugated metabolites were also detected. Five metabolites were present in sufficient amounts to quantify, using isolated urinary metabolites as reference standards. The mean fractional recovery of administered p-cymene was 0.77 ± 0.09 and 0.84 ± 0.12 for the low and high dose, respectively. The major metabolite excreted was the novel carboxy diol compound. No fecal metabolites were found. Thus, the koala employs a strategy in the metabolism of p-cymene that promotes the production of extensively oxidized metabolites that consequently have increased polarity and enhanced renal excretion. This strategy is compared with that employed by other Eucalyptus leaf folivores.     

Epicuticular Wax Chemicals in Zea mays Influence Oviposition in Ostrinia nubilalis

The chemical basis of oviposition elicitation in a generalist herbivore was determined by examination of oviposition responses in Ostrinia nubilalis to corn (Zea mays) chemicals in two-choice laboratory bioassays. A pentane extract of corn leaves stimulated oviposition and the activity persisted for three days, indicating that oviposition in O. nubilalis is elicited by low-volatility chemicals. Chemicals in the extract were fractionated by column chromatography on Florisil, using a sequence of solvents of increasing polarity. Bioassays of Florisil fractions indicated that the stimulants were eluted with nonpolar solvents. Positive bioassay results with an extract prepared by dipping corn leaves in pentane for 20 sec for extraction of leaf surface chemicals suggested that some of the active material was present in the leaf epicuticle. Gas chromatographic analyses and comparisons with retention times of standards suggested the presence of several n-alkanes in the dip extract. Five n-alkanes—hexacosane, heptacosane, octacosane, nonacosane, and tritriacontane—known to be present in the epicuticle of corn leaves were bioassayed, and all five elicited oviposition responses. These results suggest that oviposition elicitation in O. nubilalis is influenced by the presence of n-alkanes in the host plant epicuticle.