Fenton-type reactions and iron concentrations in the midgut fluids of tree-feeding caterpillars

Peroxides are formed in the midgut fluids of caterpillars when ingested tannins and other phenolic compounds oxidize. If these peroxides broke down in the presence of redox-active metal ions, they would form damaging free radicals (Fenton-type reactions). Elemental iron is present in relatively large amounts in leaves and artificial diets, but little is known about its concentration and redox state in midgut fluids, or the extent of Fenton-type reactions in these conditions. This study compared the levels of hydroxyl radicals and iron in the midgut fluids of two species of caterpillars: Orgyia leucostigma, in which phenol oxidation is limited, and Malacosoma disstria, in which phenol oxidation is more extensive. We tested two hypotheses: (1) higher levels of hydroxyl radicals are formed in M. disstria (consistent with the higher concentrations of hydrogen peroxide in this species), and (2) lower concentrations of iron are present in O. leucostigma (providing greater protection of its midgut fluids from oxidative damage). Hydroxyl radical levels increased greatly in M. disstria, but not in O. leucostigma, when they consumed a tannin-containing diet, supporting the first hypothesis. Protein oxidation was also significantly increased in the midgut fluids of M. disstria that ingested tannic acid, consistent with hydroxyl radical damage. Contrary to the second hypothesis, similar concentrations of iron (70 microM) remained in solution or suspension in both species of caterpillars on an artificial diet. Over 90% of this iron appeared to be in the reduced (catalytically active) state in both species. We conclude that tree-feeding caterpillars protect their midgut fluids from oxidative damage caused by Fenton-type reactions by limiting the formation of peroxides, rather than by limiting the availability of reduced iron.     

Semiquinone and ascorbyl radicals in the gut fluids of caterpillars measured with EPR spectrometry

The biological activity of phenolic compounds ingested by caterpillars is commonly believed to result from their oxidation, although the products of oxidation have been well-characterized in only a few cases. The initial oxidation products of phenols (semiquinone or phenoxyl radicals) can be measured with electron paramagnetic resonance (EPR) spectrometry. In this study semiquinone radicals formed from tannic acid and gallic acid in the gut fluids of two species of caterpillars were measured. In Orgyia leucostigma, in which ingested phenols are not oxidized, semiquinone radicals were absent or at very low intensities. By contrast, in Malacosoma disstria, in which ingested phenols are oxidized, high semiquinone radical intensities were measured. In the absence of detectable levels of semiquinone radicals, ascorbyl radicals were detected in the EPR spectra instead. High molar ratios of ascorbate to phenols in an artificial diet produced ascorbyl radicals in the midgut fluids of both species, while diets containing low molar ratios produced semiquinone radicals. Similar results were obtained in M. disstria fed the leaves of red oak or sugar maple. The results of this study provide further evidence that ascorbate is an essential antioxidant that prevents the oxidation of phenols in the gut fluids of caterpillars, and demonstrate that EPR spectrometry is a valuable method for determining the degree of oxidative activation of phenols ingested by herbivorous insects.     

Antioxidant defense of the midgut epithelium by the peritrophic envelope in caterpillars

The peritrophic envelope (PE) is an extracellular matrix that is secreted by the midgut epithelium in most arthropods. In addition to protecting the midgut epithelium from abrasive food particles and microbial pathogens, in vitro experiments have suggested that the PE functions as a radical-scavenging antioxidant in caterpillars. This study tested the hypothesis that the PE is a "sacrificial antioxidant" in vivo in caterpillars. As a sacrificial antioxidant, the PE would (1) bind catalytic metal ions, (2) become oxidized itself, and (3) protect the midgut epithelium from oxidative damage. Each of these functions was supported by our results: the PE in Malacosoma disstria adsorbed increased amounts of iron as the concentration of iron was increased in its diet. Iron adsorption by the PE helped protect the midgut epithelium of M. disstria from oxidative damage over a wide range of ingested iron concentrations. Secondly, while the midgut epithelium was protected, protein oxidation in the PE increased 108% when tannic acid was oxidized in the endoperitrophic space. Finally, when the formation of the PE was inhibited by Calcofluor, protein carbonyls in the midgut epithelia of M. disstria and Orgyia leucostigma increased by two- to threefold. We conclude that the PE functions as an effective iron-binding and radical-scavenging antioxidant that protects the midgut epithelia of caterpillars.     

Oxygen levels in the gut lumens of herbivorous insects

Oxygen levels were measured in the foregut and midgut lumens of ten species of caterpillars and three species of grasshoppers. In most species, the foregut was nearly anoxic, with oxygen levels ranging from 0 to 2.5 mm Hg. However, two caterpillar species with large foreguts (Malacosoma disstria and Lymantria dispar) had elevated oxygen levels (27.9 and 32.1 mm Hg) in this region when they were fed artificial diet. In all of the species surveyed, the anterior and posterior midgut were nearly anoxic, with oxygen levels ranging from 0 to 7.3 mm Hg. Oxygen levels in the midgut lumen of Helicoverpa zea did not differ when caterpillars were fed artificial diet or tomato foliage, suggesting that the insect is capable of reducing the level of ingested oxygen in its gut. An examination of the radial microgradient of oxygen in the gut lumen demonstrated that the midgut epithelium is not a sink for ingested oxygen. However, the midgut contents of larvae fed artificial diet were capable of depleting oxygen. This capacity was reduced by boiling, suggesting that the nearly anoxic state of the midgut lumen in some insects is maintained by endogenous chemical processes. We conclude that low oxygen levels in the gut lumens of most herbivorous insects may greatly reduce the rates of oxidation of ingested plant compounds by oxygen-dependent reactions.     

ALLOCATION OF CYSTEINE FOR GLUTATHIONE PRODUCTION IN CATERPILLARS WITH DIFFERENT ANTIOXIDANT DEFENSE STRATEGIES: A COMPARISON OF Lymantria dispar AND Malacosoma disstria

Sulfur amino acids [cysteine (Cys) and methionine (Met)] play two major roles during animal development: protein synthesis for growth and glutathione synthesis for defense. For caterpillars, the levels of sulfur amino acids found in foliar protein can be especially low relative to their nutritional needs. Previous work has measured concentrations of glutathione (GSH; containing Cys) in specific animal tissues, but has not examined whole‐body levels to ascertain the costliness of this defense in terms of Cys allocation. This study examined whether the production of GSH varies between species and within individuals in accordance with an insect's need for antioxidant defense. Secondly, we quantified the allocation of total Cys (peptide‐bound plus free Cys) to GSH in caterpillars as an estimate of its cost. Two contrasting species were compared: Lymantria dispar (Lymantriidae), a species that is highly defended, and Malacosoma disstria (Lasiocampidae), a species that is less defended. As expected, GSH levels were significantly higher in L. dispar than in M. disstria. Consistent with the function of the midgut as a first line of defense against ingested toxins, GSH levels were significantly higher in these tissues than in the whole bodies of both species. A major finding in this study was that a large fraction of total Cys is used to produce GSH: GSH in the midguts of L. dispar and M. disstria contained 23 and 21%, respectively, of the total Cys in these tissues, and the GSH in their remaining body tissues contained 19 and 17% of the total Cys in these tissues. Levels of total Cys in caterpillar tissues followed the same pattern of distribution as did GSH, producing a strong association between GSH and total Cys (R² = 0.794). We conclude that GSH is a costly defense, especially in generalist tree‐feeding species such as L. dispar. These results further suggest that the large allocation of Cys to GSH in highly defended species might produce a tradeoff by limiting the amount of Cys available for rapid growth.     

Glutathione–ascorbic acid redox cycle and thioredoxin reductase activity in the digestive tract of Leptinotarsa decemlineata (Say)

In view of the antioxidant role of glutathione (GSH) and ascorbic acid (AA), we have examined capacity of the GSH–AA redox cycle in relation to oxidative stress effects in the midgut of the Colorado potato beetle Leptinotarsa decemlineata. Adult gut harbors a higher capacity to cope with oxidative stress than the larval gut. Protein carbonylation was pronounced in the wall of anterior larval midgut and was generally lower in the food digest than in the gut wall. Restriction of oxidative stress effects in anterior gut lumen manifested by lipid peroxidation and protein carbonylation is interpreted as a mechanism favoring digestion and absorption in the posterior midgut. Presence of high GSH in the posterior midgut and AA in both posterior and anterior midguts of adults points to higher utility of the GSH–AA redox system in limiting oxidative stress to manageable levels. The presence, gene expression and activity of thioredoxin reductase (TrxR) were demonstrated for the first time in L. decemlineata which was markedly higher in the anterior than in the posterior midgut in both stages. It is probably central to the maintenance of reduced GSH levels in the whole gut, despite a GSSG/2GSH redox potential tending towards oxidizing ranging from ?183.5 to ?124.4 mV. Glutathione-dehydroascorbate reductase (G_DHAR) activity was markedly augmented in adult gut compared with larva, pointing to a more efficient conversion of dehydroascorbate (DHA) to AA. Also, ascorbate peroxidase (APOX) activity was significantly elevated in all gut compartments of adult except the wall of posterior midgut. The results emphasize the potential importance and role of the GSH–AA redox cycle as a defense strategy against oxidative stress in the gut of L. decemlineata.     

Stage-specific distribution of oxidative radicals and antioxidant enzymes in the midgut of Leptinotarsa decemlineata

The titers of reactive oxygen species (ROS) represented by superoxide anion and general peroxides, and the activities of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), are regulated in the midgut of the Colorado potato beetle (CPB) relative to the gut compartment, developmental stage, and food intake. ROS concentration is low in the potato leaves but it is very high in their digest in insect's anterior midgut. It is proposed that intensive ROS production in this gut region is linked to the processing of allelochemicals. SOD and CAT activities, low oxygen tension, and unidentified redox systems that maintain a slightly reducing milieu in the midgut lumen (pe+pH=6.95 declining to 5.36), obviously contribute to the decrease of ROS concentration along the gut length to a minimum in the wall of posterior midgut region. SOD and CAT activities are higher in the potato leaves than in the midgut tissues but the role of plant enzymes in ROS elimination within the gut lumen remains to be shown. A lower level of ROS and a higher antioxidant potential in the adult than in the larval midgut indicate stage specificity in the management of oxidative stress. The antioxidant defense is high in the diapausing adults that contain no detectable superoxide and about ten times less peroxides than the reproducing adults.     

Tyrosinase inhibitors from galls of Rhus javanica leaves and their effects on insects

As a defense mechanism of the leaves of Rhus javanica (Anacardiaceae) against the aphid Melaphis chinensis (Aphididae) attack, tannic acid is rapidly accumulated and forms galls along the midrib of the leaves resulting in a unique natural medicine Gallae Rhois. Tannic acid was found to inhibit the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by tyrosinase (EC 1.14.18.1) with an IC50 of 22 microM. The aphid would detoxify the ingested toxic tannic acid to relatively nontoxic gallic acid, whereas the non-adapted pink bollworm Pectinophora gossypiella larvae are sensitive to the ingested tannic acid.     

Temperature and food quality effects on growth,consumption and post-ingestive utilization efficiencies of the forest tent caterpillar Malacosoma disstria (Lepidoptera: Lasiocampidae)

Temperature and food quality can both influence growth rates, consumption rates, utilization efficiencies and developmental time of herbivorous insects. Gravimetric analyses were conducted during two consecutive years to assess the effects of temperature and food quality on fourth instar larvae of the forest tent caterpillar Malacosoma disstria Hübner. Larvae were reared in the laboratory at three different temperatures (18, 24 and 30 degrees C) and on two types of diet; leaves of sugar maple trees Acer saccharum Marsh. located at the forest edge (sun-exposed leaves) or within the forest interior (shade-exposed leaves). In general, larvae reared at 18 degrees C had lower growth rates and lower consumption rates than larvae reared at the warmer temperatures (24 and 30 degrees C). Moreover, the duration of the instar decreased significantly with increasing temperatures. Type of diet also affected the growth rates and amount of food ingested by larvae but did not affect the duration of the instar. Larvae fed sun-exposed leaves consumed more food and gained higher biomasses. Values of approximate digestibility and efficiency of conversion of ingested food were also higher when larvae were fed sun-exposed leaves. Higher growth rates with increasing temperatures were primarily the result of the shorter stadium duration. The higher growth rates of larvae fed sun-exposed leaves were possibly the result of stimulatory feeding and consequently greater food intake and also a more efficient use of food ingested. This study suggests that the performance of M. disstria caterpillars could be enhanced by warmer temperatures and higher leaf quality.     

Hydroperoxide metabolism in cyanobacteria

The enzymes involved in antioxidative activity and the cellular content of the antioxidants glutathione and ascorbate in the cyanobacteria Nostoc muscorum 7119 and Synechococcus 6311 have been examined for their roles in hydroperoxide removal. High activities of ascorbate peroxidase and catalase were found in vegetative cells of both species and in the heterocysts of N. muscorum. The affinity of ascorbate peroxidase for H2O2 was 15- to 25-fold higher than that of catalase. Increased activity of ascorbate peroxidase was observed in N. muscorum when H2O2 production was enhanced by photorespiration. Catalase activity was decreased in dilute cultures whereas ascorbate peroxidase activity increased. Ascorbate peroxidase activity also increased when the CO2 concentration was reduced. Ascorbate peroxidase appears to be a key enzyme in a cascade of reactions regenerating antioxidants. Dehydroascorbate reductase was found to regenerate ascorbate, and glutathione reductase recycled glutathione. In vegetative cells glutathione was present in high amounts (2-4 mM) whereas the ascorbate content was almost 100-fold lower (20-100 microM). Glutathione peroxidase was not detected in either cyanobacterium. It is concluded from the high activity of ascorbate peroxidase activity and the levels of antioxidants found that this enzyme can effectively remove low concentrations of peroxides. Catalase may remove H2O2 produced under photooxidative conditions where the peroxide concentration is higher.     

Redox cycling of myoglobin and ascorbate: a potential protective mechanism against oxidative reperfusion injury in muscle

Metmyoglobin catalyzes the decomposition of H2O2 as well as other hydroperoxides by using ascorbic acid as a substrate. The ratio of H2O2 reduced to ascorbate oxidized is close to one, whereas the rate of oxidation is directly proportional to both H2O2 and metmyoglobin concentrations. Ascorbate also prevents the protein modifications and the O2 evolution that accompany the reaction of metmyoglobin with hydroperoxides. In the absence of ascorbate, myoglobin and H2O2 promote the peroxidation of unsaturated fatty acids and, thus, may cause damage to cellular constituents. However, lipid peroxidation is inhibited in the presence of ascorbate and, for this reason, it is suggested that this heme protein functions in the opposite manner. The redox cycling of myoglobin by ascorbate may act as an important electron "sink" and defense mechanism against peroxides during oxidative challenge to muscle.     

Impact of dietary allelochemicals on gypsy moth (Lymantria dispar) caterpillars: importance of midgut alkalinity

Midgut pH of gypsy moth larvae was depressed artificially with buffered diet to examine the impact of alkalinity on the caterpillars' ability to tolerate a dietary polyphenol and a quinone. A 2x3 factorial design was used, with 2 levels of succinate buffer and 3 dietary amendments (tannic acid, juglone, or control). Development was monitored during the third and fourth instars, with consumption, food passage rates, midgut pH, and midgut redox potential (Eh) measured in the fourth instar. Diet buffering successfully depressed midgut pH to hypothetically suboptimal acidic levels without reductions in survivorship, but it did reduce larval growth and impede development. Buffering dramatically reduced survivorship of fourth instar larvae eating diets containing tannic acid or juglone. Growth increased on unbuffered diet amended with tannic acid, but not with juglone. Caterpillars passed food through the gut more slowly when feeding on buffered tannic acid diet or on unbuffered juglone diet. These results indicate that maintenance of midgut alkalinity is critical to tolerance of dietary tannic acid and juglone, and that these allelochemicals have very different activities in the caterpillar gut.     

Morphometry of the midgut epithelium of Diatraea saccharalis Fabricius, 1794 (Lepidoptera) parasitized by Cotesia flavipes Cameron, 1891 (Hymenoptera)

The morphometric study of the midgut in Diatraea saccharalis (Lepidoptera) larvae parasitized by the Cotesia flavipes (Hymenoptera) showed that there was significant increase in the columnar, goblet and regenerative cells and their nuclei; the midgut lumen diameter and the epithelial height were also increased in the parasitized larvae. The multivariate analysis showed that parasitism affected the columnar cell only in the posterior region, and the goblet cells along the midgut length (anterior and posterior regions).     

Permeability of the Peritrophic Envelopes of Herbivorous Insects to Dextran Sulfate: a Test of the Polyanion Exclusion Hypothesis

We tested the hypothesis that the permeability of the peritrophic envelope in herbivorous insects is greatly reduced for polyanions as a result of an extensive network of anionic sites in the proteoglycans of the matrix. 14C-Dextran sulfate (polyanionic, 8000 M(w)) and fluorescein isothiocyanate-labeled (FITC) dextran (monoanionic, 9400 M(w)) were introduced together into the endoperitrophic space of the midguts of Orgyia leucostigma (Lepidoptera) larvae and Melanoplus sanguinipes (Orthoptera) adults. In all cases more of the 14C-dextran sulfate permeated the peritrophic envelope than the FITC-dextran, the opposite of the result predicted by the polyanion exclusion hypothesis. We conclude that polyanion exclusion is not a mechanism that contributes significantly to the permeability properties of the peritrophic envelopes of these two species, or that explains the failure of tannic acid to cross the peritrophic envelopes of lepidopteran larvae. Copyright 1997 Elsevier Science Ltd. All rights reserved     

Formation of insoluble and colloidally dispersed tannic acid complexes in the midgut fluid of Manduca sexta (Lepidoptera: Sphingidae): An explanation for the failure of tannic acid to cross the peritrophic envelopes of lepidopteran larvae

Magnesium and calcium ions, in concentrations comparable to those reported in the midgut fluids of lepidopteran larvae, bring about the precipitation of most of the tannic acid present in simple solutions buffered at pH 8.0 and 10.0, but not at pH 6.5. In contrast, when tannic acid is added to Manduca sexta midgut fluid, less than 31% of the tannic acid added to the gut fluid is converted to a form that can be centrifuged into a pellet. The rest remains in the supernatant solution in the form of a colloidal suspension. Very little of the tannic acid, if any, remains in true solution. We suggest that the tannic acid-containing phase that is produced when tannic acid is added to midgut fluid is a complex multi-molecular aggregate of indefinite chemical composition, incorporating varying amounts of tannic acid, surface-active phospholipids, proteins, and polyvalent metal ions. On the basis of this study, we further suggest that the failure of tannins to diffuse across the peritrophic envelopes of lepidopteran larvae is a result of the capacity of the peritrophic envelope to act as a physical barrier to insoluble and colloidally dispersed particles, not the presence of substances in the matrix that strongly adsorb polyphenols or the presence of an extensive network of fixed anionic sites in the matrix that acts as an electrostatic barrier to the passage of polyphenolate anions. Arch. Insect Biochem. Physiol. 39:109–117, 1998.© 1998 Wiley-Liss, Inc.     

Selective secretion of free glycine,a neutralizer against a plant defense chemical,in the digestive juice of the privet moth larvae

The larva of the privet moth, Brahmaea wallichii (Brahmaeidae) is a specialist feeder of the privet tree, Ligustrum obtusifolium (Oleaceae). A very high concentration (50 mM or 0.4%) of free glycine, found in the digestive juice of the larvae, works as a neutralizer against the very strong protein-denaturing activity of privet leaves that is caused by oleuropein, an iridoid that functions in chemical defense. Concentration of free glycine was high in the anterior region of the midgut lumen and low in the posterior region. To examine if some glycine-specific secretion mechanism exists, injection experiments were performed using (15)N-labeled amino acids. When 13 &mgr;mol (1 mg) of (15)N-glycine was injected into hemolymph of fifth instar larvae of B. wallichii, a high concentration of (15)N (5 mM or 75 &mgr;g/g midgut content) was detected in the anterior parts of the midgut lumen 1 h after injection. (15)N-NMR data indicated at least 60% of the (15)N found in midgut lumen existed as (15)N-glycine. Approximately, 25% of the injected (15)N-glycine was estimated to have moved from the hemolymph to the midgut lumen. In contrast, no (15)N was detected in the midgut lumen when 13 &mgr;mol of (15)N-labeled alanine, lysine and glutamate were injected into hemolymph. Glycine was the only amino acid whose concentration was higher in the midgut lumen (50 mM) than in the hemolymph (22 mM). These data suggest the existence of some active and glycine-specific secretory mechanism in the midgut of B. wallichii.     

Alkalization of larval mosquito midgut and the role of carbonic anhydrase in different species of mosquitoes

We have previously demonstrated the involvement of carbonic anhydrase (CA) in the alkalization mechanism of the Aedes aegypti larval midgut. In this study, we used Hansson's histochemistry to examine the distribution of the enzyme in the midgut of six different species of mosquito larvae (Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, Culex nigripalpus, Ochlerotatus taeniorhynchus, Anopheles quadrimaculatus). Additionally, we quantitated CA content in the gastric caeca, anterior and posterior midgut of fourth instar larvae from these species using the 18O isotope exchange method coupled to mass spectrometry. We also tested the effect of CA inhibitors such as methazolamide and acetazolamide in the alkalization of the midgut for these species. Our results indicate that CA is present in the larval midgut of the species studied and that it appears to be associated with the posterior midgut and gastric caeca in some species and with the anterior midgut in others. CA inhibitors appear to have a profound effect on the alkalization mechanism of the midgut with lethal consequences for most of the species tested.     

Histopathological effects of tannic acid on the midgut epithelium of some aquatic Diptera larvae

The impact of tannins on larval Nematocera was investigated by an extensive survey of the relative toxicity of tannic acid in Diptera larvae representative of mosquito communities from alpine hydrosystems (Culicidae, Chaoboridae, Chironomidae, and Simuliidae) together with a nonindigenous vector competent Culicidae species. Bioassays indicate that exposure to tannic acid at concentrations from 0.25 to 4 mM is deleterious for Culex pipiens, Simulium variegatum, and Chironomus annularius, but not for Aedes, Anopheles, Culiseta, and Chaoborus species. Histopathological observations reveal that, among the target organs of tannic acid, mainly the midgut epithelium is affected by treatment. However, the extent of degeneration varies according to the taxon, the duration of the treatment, and the concentrations assayed. The vulnerability of epithelial cells differs among cell types, clear cells of the anterior midgut showing symptoms of intoxication before dark cells of the posterior midgut. The toxic effects of tannic acid are discussed, particularly in comparison to those of insecticidal bacteria, in order to evaluate the potential for use of tannins in the regulation of larval populations of dipteran pests.     

Ascorbate peroxidase: A novel antioxidant enzyme in insects

Ascorbate peroxidase (APOX) activity, which catalyzes the oxidation of ascorbic acid with the concurrent reduction of hydrogen peroxide (H₂O₂), was found in larvae of Helicoverpa zea. Since insects apparently lack a Se-dependent glutathione peroxidase and since catalase has a low affinity for H₂O₂, this enzyme may be important in removing H₂O₂ in insects. We partially purified the APOX activity 58x from the whole body homogenates and investigated its activity with model lipid peroxides, electron donors, and known inhibitors of plant APOX. The H. zea APOX has activity with model lipid peroxides. This, along with the APOX activity found in fat body tissues, suggests that ascorbate peroxidase may be important in removing lipid peroxides in insects. The H. zea APOX has broader specificity for electron donors than the plant APOX with activity using cysteine, NADPH, glutathione, and cytochrome C as electron donors (22–93% of activity with ascorbate). The H. zea APOX is also resistant to many of the known inhibitors of plant APOX, suggesting that the enzyme has a different active site and may not be a heme-peroxidase. © 1997 Wiley-Liss, Inc.