Tannin sensitivity in larvae ofMalacosoma disstria (Lepidoptera): Roles of the peritrophic envelope and midgut oxidation

Final-instarMalacosoma disstria fed artificial diets containing tannic acid develop lethal pupal deformities. We examined some of the factors potentially underlying tannin sensitivity in this species, including the permeability of the peritrophic envelope to tannic acid and the chemical fate of tannic acid in the gut. Tannic acid does not penetrate the peritrophic envelope ofM. disstria, demonstrating that the containment of tannic acid within the endoperitrophic space is not sufficient to protect an insect herbivore from the adverse effects of ingested tannins. Ingested tannic acid undergoes extensive chemical modification in the midgut. Only 19–21 % of the high molecular weight components of the tannic acid ingested was recovered in the frass. Of two possible chemical fates of ingested tannic acid, oxidation is the predominant chemical transformation, whereas little hydrolysis occurs. Measurements of gut redox parameters showed that conditions in the midgut favor the oxidation of phenols. However, similar conditions occur in the midguts ofOrgyia leucostigma, in which no oxidation occurs. Therefore, oxidizing gut redox conditions do not necessarily lead to polyphenol oxidation in lepidopteran larvae. We conclude that the sensitivity ofM. disstria to ingested tannins is a consequence of their oxidation in the midgut.     

Gut-based antioxidant enzymes in a polyphagous and a graminivorous grasshopper

Graminivorous species of grasshoppers develop lethal lesions in their midgut epithelia when they ingest tannic acid, whereas polyphagous grass- hoppers are unaffected by ingested tannins. This study tests the hypothesis that polyphagous species are defended by higher activities of antioxidant enzymes (constitutive or inducible) in their guts than are graminivorous species. Comparisons were made between four antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APOX), and glutathione transferase peroxidase (GSTPX). Enzyme activities were measured in the gut lumens and midgut tissues of Melanoplus sanguinipes (polyphagous) and Aulocara ellioti (graminivorous). The results of this study do not support the hypothesis that M. sanguinipes is better defended by antioxidant enzymes than is A. ellioti, nor are these enzymes more inducible in M. sanguinipes than in A. ellioti when insects consume food containing 15% dry weight tannic acid. Instead, tannic acid consumption reduced SOD, APOX, and GSTPX activities in both species. This study reports the first evidence that SOD is secreted into the midgut lumen in insects, with activities two- to fourfold higher than those found in midgut tissues. The spatial distribution of GSTPX and APOX activities observed in both species suggests that ingested plant antioxidant enzymes may function as acquired defenses in grasshoppers. In addition, the results of this study permit the first comparison between the antioxidant enzyme defenses of Orthoptera and Lepidoptera. Most notably, grasshoppers have higher SOD activities than caterpillars, but completely lack APOX in their midgut tissues.     

Plant Secondary Compounds and Grasshoppers: Beyond Plant Defenses

Modern grasshoppers probably evolved from polyphagous ancestors endowed with the ability to tolerate many plant secondary compounds. This tolerance involves various behavioral and anatomical adaptations. Polyphagous grasshoppers have a relatively low level of sensitivity to the taste of many secondary compounds, and, if they do respond to the taste, have the capacity to habituate. This gives time for the induction of detoxifying enzymes so that unpalatable but potentially nutritious plants may be eaten safely. Associative learning involving secondary compounds may be important in food aversion learning, enabling the insects to avoid foods that have inappropriate nutrients, for example. Learning is also involved when grasshoppers develop associations between the taste of chemicals in the surface waxes of plants and internal leaf chemistry, enabling them to make faster decisions about the acceptability of a plant. Anatomically, the midgut ceca of polyphagous grasshoppers have well-developed posterior arms, and it is possible that these are especially important in detoxification, while some species, in addition, have a specialized pocket region in which macromolecules accumulate to be eliminated from the body when the lining of peritrophic envelope is drawn out. Polyphagous species also have thick peritrophic envelopes to which various phenolics become adsorbed. Finally, the midgut environment contains surfactants that reduce tannin–protein complexing except at very high tannin concentrations. Some polyphagous species can utilize secondary compounds as defensive substances or, in one case, in cuticular sclerotization. Grass feeding has evolved on numerous occasions from these polyphagous ancestors, and it has been associated with a loss of many of the characters providing protection from secondary compounds.     

Antioxidants in grasshoppers: higher levels defend the midgut tissues of a polyphagous species than a graminivorous species

Polyphagous grasshoppers consume plants that contain markedly greater amounts of potentially prooxidant allelochemicals than the grasses eaten by graminivorous grasshoppers. Therefore, levels of antioxidant defenses maintained by these herbivores might be expected to differ in accordance with host plant ranges. Antioxidant levels were compared in midgut tissues and gut fluids of a polyphagous grasshopper, Melanoplus sanguinipes, and a graminivorous grasshopper, Aulocara ellioti. Glutathione concentrations in midgut tissues of M. sanguinipes (10.6 mM) are among the highest measured in animal tissues and are twice as high as those in A. ellioti. -Tocopherol levels are 126% higher in midgut tissues of M. sanguinipes than in those of A. ellioti, and remain at high levels when M. sanguinipes is reared on plants containing a wide range of -tocopherol concentrations. Ascorbate levels in M. sanguinipes midgut tissues are 27% higher than in those of A. ellioti, but vary depending on the host plant on which they are reared. Midgut fluids of both species contain elevated levels of glutathione, as well as large (millimolar) amounts of undetermined antioxidants that are produced in the insects. The consumption of tannic acid decreases ascorbate concentrations in midgut tisssues and gut fluids of A. ellioti but has no effect on ascorbate levels in M. sanguinipes. The results of this study provide the first measurements of antioxidants in grasshoppers and suggest that the maintenance of high levels of antioxidants in the midgut tissues of polyphagous grasshoppers might effectively protect them from oxidative stress.     

The Fate and Effects of Ingested Hydrolyzable Tannins in Porcellio scaber

When adults of Porcellio scaber fed on litter prior to an artificial diet containing 5% of commercially available tannic acid, 55% of the ingested galloylglucose esters was excreted unchanged, about 25% was hydrolyzed, and 20% was oxidized during the gut passage. After reducing the counts of microorganisms in the gut of P. scaber, the data obtained indicated an important role of ingested platable microorganisms in hydrolyzing gallotannins. Oxidation of phenolics appeared to be mainly due to the endosymbiotic bacteria of the hepatopancreas. Microbial counts in the hindgut were strongly reduced by ingested galloylglucose esters, while gallic acid in the diet (2%) reduced the number of palatable fungi and bacteria less strikingly, and increased the total number of the gut microbiota. Hepatopancreatic bacteria were only slightly affected by ingested tannic acid, since the hepatopancreas contained only few galloylglucose esters. This may be due to the permeability of the hindgut cuticle: the cuticle of the anterior hindgut was freely permeable to gallic acid, while it was nearly impermeable to larger polyphenols. The cuticle of the posterior hindgut was permeable to only about 4% of the gallic acid present in the hindgut lumen. The results are discussed with respect to potentially harmful effects of ingested hydrolyzable tannins and their digestion in Porcellio scaber.     

Failure of tannic acid to inhibit digestion or reduce digestibility of plant protein in gut fluids of insect herbivores

The rate of hydrolysis of the abundant foliar protein, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPC), in enzymatically active gut fluid ofManduca sexta larvae is very rapid and is unaffected by the presence of tannic acid, even when tannic acid is present in the incubation mixture in amounts in excess of the amount of RuBPC. When this protein is dissolved in the denatured gut fluids ofM. sexta larvae orSchistocerca gregaria nymphs, large amounts of tannic acid must be added to bring about the precipitation of significant quantities of protein. The ability of insect gut fluid to prevent the formation of insoluble tannin-protein complexes is due to the presence of surfactants. On the basis of our results and a review of the findings of other investigators, we argue that there is no evidence that tannins reduce the nutritional value of an insect's food by inhibiting digestive enzymes or by reducing the digestibility of ingested proteins and, further, that the failure of tannins to interfere with digestion is readily explained on the basis of well-documented characteristics of the digestive systems of herbivorous insects. In challenging the currently popular notion that tannins are digestibility-reducing substances, we do not challenge the general utility of either the apparency theory or resource availability theory of plant defense. In debating the merits of these two analyses of plant-herbivore interactions, however, the demise of tannins as all-purpose, dose-dependent, digestibility-reducing defensive substances must be taken into account.     

Effects of Plant Diet on Detoxification Enzyme Activities of Two Grasshoppers, Melanoplus differentialis and Taeniopoda eques

The polyphagous grasshoppers Melanoplus differentialis and Taeniopoda eques use different foraging patterns: over time M. differentialis tends to reduce the variety of host plants it feeds on and specialize on particular plants (diet components), whereas T. eques mixes host plants to achieve a very diverse diet. We tested the hypothesis that these differing behaviors are correlated with differing patterns of detoxification enzymes. The activities of midgut, fat body, and malpighian tubule detoxification enzymes were determined in last instars of the two grasshoppers, reared for five days on single-or mixed-plant diets. Significant differences in several cytochrome P450 activities and glutathione S-transferase were evident for nymphal grasshoppers feeding on different plant diets. However, the behavioral differences between the two species could not be explained by an underlying flexibility of detoxification response in M. differentialis, but lacking in T. eques. This is the first reported evidence that detoxification enzyme activities are affected by plant diet in polyphagous orthopterans.     

Evaluating Ascorbate Oxidase as a Plant Defense Against Leaf-Chewing Insects Using Transgenic Poplar

Ascorbate is the major water-soluble antioxidant in plants and animals, and it is an essential nutrient for most insect herbivores. Therefore, ascorbate oxidase (AO) has been proposed to function as a plant defense that decreases the availability of ascorbate to insects. This hypothesis was tested by producing transgenic poplar (Populus tremula × Populus alba; Salicaceae) with 14- to 37-fold higher foliar AO activities than control (wild type) leaves and feeding these leaves to Lymantria dispar L. (Lepidoptera: Lymantriidae) caterpillars and Melanoplus sanguinipes (Fabricius) (Orthoptera: Acrididae) grasshoppers. To examine potential mechanisms of activity of AO in these insects, ascorbyl radical and/or ascorbate levels were measured in gut contents. No significant changes in ascorbyl radical or ascorbate levels were found in the midgut contents of L. dispar larvae that ingested the leaves of the AO-overexpressing genotypes compared to the control genotype, and no significant decreases in ascorbate levels were found in the foregut or midgut contents of M. sanguinipes. Treatment of control leaves with commercial AO also produced no changes in the midgut biochemistry of L. dispar larvae, as measured by levels of ascorbyl radicals. Likewise, no increase in oxidative stress was observed in L. dispar that consumed tannin-treated AO-overexpressing leaves compared with tannin-treated control genotype leaves. Performance experiments were carried out on first- and fourth-instar L. dispar larvae on leaf disks and on third instars feeding on intact leaves on trees. In no case was a significant difference found in the contrast between the control and three AO-overexpressing genotypes for relative consumption rate, relative growth rate, or nutritional indices. We conclude that elevated levels of AO in poplar are unlikely to serve as a defense against herbivores such as L. dispar or M. sanguinipes and that the low oxygen levels commonly found in the guts of caterpillars and grasshoppers may limit the activity of ingested AO in these leaf-chewing insects.     

Phlorotannin-protein interactions

Tannins are one of the most broadly distributed types of plant secondary compounds, and have been the focal point for many studies of plant/herbivore interactions. Tannins interact strongly with proteins, so that the fate and effects of ingested tannins are in part dependent on the mode of interaction of the tannin with dietary and endogenous proteins in an herbivore's gut. We investigated the factors affecting the precipitation of proteins by phlorotannins from three species of marine brown algae:Carpophyllum maschalocarpum, Ecklonia radiata, andLobophora variegata. Phlorotannins were precipitated by proteins in a pH-dependent and concentration-dependent fashion. Precipitation also varied as a function of the presence of reducing agent, the type of phlorotannin or protein used, and the presence of organic solvents such as hydrogen bond inhibitors. Of particular significance was the ability of some phlorotannins to oxidize and form covalent bonds with some proteins. In contrast, under similar experimental conditions three types of terrestrial tannins (procyanidins, profisetinidins, and gallotannins) apparently did not form covalent complexes with proteins. Our results suggest several ways in which the biological activity of phlorotannins may vary as a function of the properties of the gut environment of marine herbivores. Moreover, we identify specific structural characteristics of phlorotannins which affect their tendency to oxidize, and thus, their potential effects on marine herbivores.     

Chemical aspects of host-plant specificity in threeLarrea-feeding grasshoppers

The host-selection behavior of three species of grasshopper feeding on creosote bush,Larrea tridentata, in southern California was investigated. The species wereBootettix argentatus, which is monophagous;Ligurotettix coquilletti, oligophagous; andCibolacris parviceps, polyphagous. The monophagous species is stimulated to bite by nordihydroguaiaretic acid (NDGA), a compound that is characteristic of the host plant and that may comprise up to 10% of the dry weight of the leaf. Host specificity ofB. argentatus is enhanced by deterrent responses to compounds present in the surface waxes of all non-host-plant species. Both the oligophagous and polyphagous species are deterred by NDGA at naturally occurring concentrations. Their association withLarrea is probably based on tolerance of the plant chemicals rather than on dependence on specific chemicals. Factors other than the chemistry of the plant probably also contribute to the specificity ofB. argentatus andL. coquilletti.     

Feeding behavior of graminivorous grasshoppers in response to host-plant extracts,alkaloids, and tannins

Secondary metabolites exhibit the potential to direct food selection by grass-feeding (graminivorous) grasshoppers. We examined the effects of plant extracts and representative secondary metabolites on the feeding behavior of two such grasshoppers,Ageneotettix deorum (Scudder) andPhoetaliotes nebrascensis (Scudder). Three alkaloids and two tannins were bioassayed for their activity as feeding deterrent allelochemicals, as were extracts from the foliage of the graminoids commonly eaten by these grasshoppers:Agropyron smithii Rydb.,Andropogon hallii Hack.,Andropogon scoparius Michx.,Bouteloua gracilis (H. B. K) Lag. ex Griffiths,Carex heliophila Mack. andStipa comata Trin. & Rupr. Alkaloids strongly deterred feeding but tannins only exhibited a weak effect, even when present at four times the concentration of total phenolics typical for these graminoids. Host-plant extracts also exhibited weak effects, such that we found no evidence for either strong deterrence or phagostimulation. Our results for alkaloids and host-plant extracts are consistent with the view that grass-feeding grasshoppers may be restricted to graminoids because of: (1) the presence of deterrents in nonhosts and (2) the absence of deterrents in hosts. However, our data for tannins show that these are unlikely to be effective barriers to herbivory by these grasshoppers.     

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.     

Olfactory responses of a specialist and a generalist grasshopper to volatiles ofArtemisia ludoviciana nutt. (Asteraceae)

Hypochlora alba is a specialist grasshopper that lives and feeds almost exclusively onArtemisia ludoviciana, a plant that produces large amounts of allelochemics including a variety of monoterpenes. This plant is not a host for generalist grasshoppers such asMelanoplus sanguinipes. The role of olfaction in the grasshopper-plant relationship was investigated by comparing electroantennograms (EAGs) of males and females of both species generated by solvent-extracted volatiles from plant leaves and by major individual components. Volatiles ofA. ludoviciana were identified by gas chromatography-mass spectrometry. The major components were 1,8-cineole, camphor, camphene, and borneol, while minor identified compounds were - and -pinene, -thujene, myrcene,p-cymene, Artemisia ketone, -thujone, and bornyl acetate. The EAGs (mV) ofH. alba males to a range of concentrations of individual volatiles or the total plant extract were nearly double those of conspecific females or both sexes ofM. sanguinipes. However, both sexes ofM. sanguinipes were more sensitive than either sex ofH, alba to geraniol, a monoterpene that commonly occurs in many plant species but is absent or is present in only trace amounts inA. ludoviciana. The increased sensitivity ofH. alba males to the odor components of their host plant appears to be related to the greater number of certain olfactory chemoreceptors on male versus female antennae. The significance of this phenomenon is discussed.     

Differential Sensitivity of Mosquito Taxa to Vegetable Tannins

The sensitivity of larval Culicidae to vegetable tannins was investigated in different taxa representative of the fauna from alpine hydrosystems (Aedes rusticus, Culex pipiens) and foreign noxious fauna (Aedes aegypti, A. albopictus). Bioassays reveal that tannic acid at concentrations of 0.1–6 mM is significantly more toxic for C. pipiens than for Aedes taxa, and A. aegypti is more sensitive than A. albopictus and A. rusticus. Comparison of the rank order of sensitivity among taxa with the associated levels of cytochrome P-450, esterase, and glutathione-S-transferase activities suggests that cytochrome P-450 and esterases may be involved in the detoxification of tannins. A possible involvement of these detoxifying enzymes is also revealed in vivo by the synergistic effects of S,S,S-tributyl phosphorotrithioate (esterase inhibitor) and piperonyl butoxide (P-450 inhibitor). The differential sensitivity to tannins among taxa is discussed in terms of ecological implications within mosquito communities from alpine hydrosystems, where the acquisition of tannins–detoxifying enzymatic systems may be considered as a key innovation.     

Influence of oleoresin constituents fromPinus ponderosa andPinus jeffreyi on growth of mycangial fungi fromDendroctonus ponderosae andDendroctonus jeffreyi

Dendroctonus jeffreyi andD. ponderosae are morphologically similar sympatric species of pine bark beetles over portions of their geographic ranges; however,D. jeffreyi is monophagous onP. jeffreyi whileD. ponderosae is highly polyphagous. Both species carry a species of mycangial fungi that are also very similar in appearance. Growth of the two mycangial fungi and of the fungusLeptographium terebrantis (associated with the polyphagous and non-tree-killingDendroctonus valens) in the presence of oleoresin constituents of host and nonhost conifers was tested by placing individual chemicals on agar growth medium and by growing the cultures in saturated atmospheres of the chemicals. The fungus associated withD. jeffreyi showed greater tolerance for chemical constituents placed on the growth medium than the other two fungi, and growth after three days was enhanced by heptane, the dominant constituent ofP. jeffreyi oleoresin. Growth of all three species of fungi was reduced by the resin constituents when the chemicals were presented as saturated atmospheres. The results suggest that the influence of the tree on growth of the symbiotic fungi of the bark beetles during the initial attack process may be different than after colonization by the beetles is complete. The difference in the responses of the apparently related species of mycangial fungi may provide some new insight into the evolutionary history of these beetle/mycangial fungus/host tree systems.     

Effects of elevated CO<Subscript>2</Subscript> on foliar quality and herbivore damage in a scrub oak ecosystem

Atmospheric CO₂ concentrations have increased exponentially over the last century and continuing increases are expected to have significant effects on ecosystems. We investigated the interactions among atmospheric CO₂, foliar quality, and herbivory within a scrub oak community at the Kennedy Space Center, Florida. Sixteen plots of open-top chambers were followed; eight of which were exposed to ambient levels of CO₂ (350 ppm), and eight of which were exposed to elevated levels of CO₂ (700 ppm). We focused on three oak species, Quercus geminata, Quercus myrtifolia, Quercus chapmanii, and one nitrogen fixing legume, Galactia elliottii. There were declines in overall nitrogen and increases in C:N ratios under elevated CO₂. Total carbon, phenolics (condensed tannins, hydrolyzable tannins, total phenolics) and fiber (cellulose, hemicellulose, lignin) did not change under elevated CO₂ across plant species. Plant species differed in their relative foliar chemistries over time, however, the only consistent differences were higher nitrogen concentrations and lower C:N ratios in the nitrogen fixer when compared to the oak species. Under elevated CO₂, damage by herbivores decreased for four of the six insect groups investigated. The overall declines in both foliar quality and herbivory under elevated CO₂ treatments suggest that damage to plants may decline as atmospheric CO₂ levels continue to rise.     

Differential effects of condensed and hydrolyzable tannin on polyphenol oxidase activity of attine symbiotic fungus

The leaf-cutting antAtta cephalotes is a generalist herbivore of the neotropics and collects leaf material to cultivate a fungus. It appears that this fungus, a Basidiomycete, is responsible for the ability of the ants to utilize most of the available woody plant species. Tannins and other phenolics are ubiquitous secondary chemicals in woody plants, and Basidiomycete fungi produce enzymes, such as polyphenol oxidase, that are capable of polymerizing and inactivating the phenolics. This study evaluates the effects of a condensed and a hydrolyzable tannin on the activity of polyphenoi oxidase and the growth of the fungus. I hypothesized that low concentrations of tannin would not inhibit polyphenol oxidase activity but high concentrations would inhibit the enzyme. Consequently, I predicted that only high concentrations of tannin would inhibit fungal growth. Laboratory assays with the fungus indicated that hydrolyzable tannin (tannic acid) and condensed tannin (quebracho tannin) differ in the mechanism of inhibition. Tannic acid does not inhibit polyphenol oxidase activity but does inhibit fungal growth. Quebracho tannin, however, inhibits both polyphenol oxidase activity and fungal growth. As predicted, both tannic acid and quebracho tannin primarily inhibit the fungus at high concentrations.     

Do defoliation and subsequent phytochemical responses reduce future herbivory on oak trees?

Perennial plants are thought to respond to partial or complete defoliation by producing new foliage that is less susceptible to herbivores because of induction of allelochemicals. Here, I tested this hypothesis by manually removing primary foliage from branches ofQuercus emoryi (Fagaceae) at two different times in the season and monitoring changes in protein and tannin levels and the amount of herbivory relative to control branches. New, secondary leaves had 2.5 × greater hydrolyzable tannin content than mature foliage of control branches. Condensed tannins, which constitute a relatively low fraction of leaf mass, were lower, while protein content was temporarily greater, in new secondary leaves relative to mature leaves. Despite large increases in hydrolyzable tannins, herbivory levels were greater on refoliated branches than on control branches. New foliage is susceptible to herbivory regardless of when it is produced in the season, possibly because lower toughness and higher water content override any induced or developmentally related changes in allelochemistry. My results do not support the hypothesis that postherbivore changes in phytochemistry protect perennial plants from future herbivory, at least within a growing season.