Metabolism and excretion of the furanocoumarin xanthotoxin by parsnip webworm,Depressaria pastinacella

The parsnip webworm,Depressaria pastinacella, feeds on plants containing high concentrations of furanocoumarins. compounds toxic to many organisms. Parsnip webworm larvae were fed radiolabeled xanthotoxin to quantify the detoxification of this furanocoumarin. They metabolized approximately 95% of the ingested xanthotoxin, indicating that metabolic detoxification is important in their tolerance to this allelochemical. Excretion of xanthotoxin and its metabolites was not restricted to the frass but also occurred by means of the silk glands. The silk glands contained half as much of the tritiated compounds as the rest of the body. Because of the feeding habits of this insect, such an excretory pathway may have implications for interactions with predators and pathogens.     

Effects of furanocoumarins on feeding behavior of parsnip webworms Depressaria pastinacella

The parsnip webworm, Depressaria pastinacella, exhibits limited physiological resistance to furanocoumarin toxins in its principal host, the wild parsnip, Pastinaca sativa. These insects are typically found attacking individual plants low in furanocoumarins, relative to others within populations. They also feed preferentially on parthenocarpic fruits, which are lower in furanocoumarin content than are normal fruits. However, in a previous study with artificial diets, they did not appear to discriminate between high and low concentrations of furanocoumarins. In this study, the ability of webworms to distinguish between diets differing in furanocoumarin content was examined with an artificial diet containing wild parsnip and in green parsnip fruits with and without supplemental furanocoumarins. Larvae showed no preference for high or low furanocoumarin diets containing equal amounts of freeze-dried parsnip fruit powder. When given a choice between otherwise similar wild parsnip fruits, webworms strongly preferred fruits that were not augmented with furanocoumarins in one plant but showed no preference or only a weak preference for nonaugmented fruits in four other plants. In order to identify chemical constituents other than furanocoumarins that might determine feeding preferences, we compared the chemical profile of parthenocarpic fruits (which are preferred) to that of normal fruit. Octyl butyrate, a known deterrent to webworms, is highly correlated with furanocoumarin content, occurs in all plants, and differs significantly among normal and parthenocarpic fruit, suggesting that webworms may be able to avoid furanocoumarins by virtue of their behavioral response to octyl butyrate.     

Behavioral responses of the parsnip webworm to host plant volatiles

Sixth instars of the parsnip webworm, Depressaria pastinacella, orient by olfaction to bud, male flowers, and female flowers of their primary host plant, Pastinaca sativa. Because octyl acetate and octyl butyrate are characteristic of tissues consumed by the sixth instar, we investigated the influence of these esters on webworm feeding behavior and chemo-orientation. Although octyl acetate and octyl butyrate are feeding deterrents, and octyl butyrate is an olfactory repellent, octyl acetate serves as an olfactory attractant. In olfactometers, webworms do not show a preference when given a choice between octyl acetate and host plant tissues. These findings suggest that octyl acetate is a sufficient cue for olfactory orientation. Such behavior may explain differences in the relative abundance of these esters observed among populations of wild parsnip under differential selection pressure from these insects.     

Characterization of furanocoumarin metabolites in parsnip webworm,Depressaria pastinacella

Although metabolites of furanocoumarins have been characterized in a wide range of organisms, to date they have been identified in only a single insect species, Papilio polyxenes. Depressaria pastinacella, the parsnip webworm, like P. polyxenes a specialist on Apiaceae, routinely consumes plant tissues higher in furanocoumarin content than does P. polyxenes and is capable of faster cytochrome P-450-mediated detoxification of these compounds. In this study, we characterized metabolites of xanthotoxin, a linear furanocoumarin, and sphondin, an angular furanocoumarin, in midguts and frass of parsnip webworms. Two metabolites were isolated and identified from webworms fed artificial diet containing xanthotoxin. LC-ESI-MS analysis resulted in the determination of a MW of 266 for the compound in the frass and one of the compounds in the midgut; ¹H NMR confirmed its structure as 6-(7-hydroxy-8-methoxycoumaryl)-hydroxyacetic acid (HCHA). The second compound from the midgut had a MW of 252 and was identified by ¹H NMR and ¹³C NMR analysis as 6-(7-hydroxy-8-methoxycoumaryl)-hydroxyethanol) (HMCH). Whereas HCHA has been found in frass of Papilio polyxenes fed xanthotoxin, HMCH has not been reported previously in insects. Although the first step of metabolism of xanthotoxin in webworms as well as P. polyxenes is likely the formation of an epoxide on the furan ring, angular furanocoumarin metabolism in webworms appears to differ. The principal metabolite of sphondin was identified as demethylated sphondin (6-hydroxy-2H-furo[2,3-h]-1-benzopyran-2-one) by LC-ESI-MS and confirmed by ¹H NMR and ¹³C NMR analyses. That webworms produce metabolites of xanthotoxin in common not only with other Lepidoptera (e.g., HCHA) but with other vertebrates (e.g., HMCH) suggests a remarkable conservatism in the metabolic capabilities of cytochrome P-450s and raises the possibility that insects may share other detoxification reactions with vertebrates with respect to toxins in foodplants.     

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.     

Behavioral Effects of Carotenoid Sequestration by the Parsnip WebwormDepressaria pastinacella

The parsnip webwormDepressaria pastinacella, acquires a distinct yellow stripe when it consumes the yellow flowers of its principal host plantPastinaca sativa, the wild parsnip. Caterpillars raised on artificial diet lacking host-plant material lack this yellow coloration. By chemical characterization and comparison of caterpillars raised on parsnip flowers and on artificial diet, we were able to determine that lutein, along with smaller amounts of other xanthophylls from the host plant, is selectively sequestered in the fat body. In bioassays designed to measure avoidance of ultraviolet light, caterpillars raised on parsnip flowers or on artificial diet supplemented with lutein were less likely to avoid exposure to ultraviolet light than caterpillars raised on unaugmented artificial diet and thus lacking sequestered carotenoids. The ability to sequester xanthophylls, which are highly effective antioxidants, may confer a selective advantage on these caterpillars, whose apiaceous host plants produce large quantities of furanocoumarins, natural products that are photoactivated by light wavelengths in the ultraviolet region; such sequestered pigments may reduce not only the oxidative stress associated with ultraviolet light and diurnal foraging but also the photooxidative stress associated with ingestion of photoactive furanocoumarins.     

Toxicity of linear furanocoumarins toSpodoptera exigua: Evidence for antagonistic interactions

The linear furanocoumarins psoralen, bergapten, and xanthotoxin were tested for toxicity to the beet armywormSpodoptera exigua (Hübner) under short ultraviolet (UVB) radiation. Increased dietary concentrations of each furanocoumarin significantly decreased insect larval weight, extended generation time, and induced higher mortality. Xanthotoxin was the most toxic, followed by psoralen and bergapten. Combining psoralen with bergapten, xanthotoxin, or both resulted in significantly antagonistic effects on insect mortality. The combination of bergapten and xanthotoxin, however, produced additive effects. The implications of these observations forS. exigua resistance in the wild plant accession ofApium prostratum and the enigma the findings represent for plant-insect relationships are discussed.     

Impact of UV radiation on activity of linear furanocoumarins andBacillus thuringiensis var.Kurstaki againstSpodoptera exigua: Implications for tritrophic interactions

Acidic fogs with a pH of 2.0 and duration of 2 hr did not reduce the efficacy ofBacillus thuringiensis var.Kurstaki (Berliner). Therefore, the impact of UV radiation was investigated on the interactions between (1) levels of the antibacterial linear furanocoumarins psoralen, bergapten, and xanthotoxin inApium graveolens (L.) occurring following a 2.0 pH acidic fog episode, (2) the noctuidSpodoptera exigua (Hübner), and (3) a sublethal dosage of the microbial pathogenB. thuringiensis var.Kurstaki. Mean time to pupation in the absence of UV radiation (survival was too low to conduct this analysis for insects exposed to UV) was significantly extended by the addition of either psoralens orB. thuringiensis. Larvae developing on diets containingB. thuringiensis plus psoralens required nearly 40% longer to pupate than controls, but their effects were additive as the interaction was not significant. Although the mean times to adult emergence were significantly different, time spent in the pupal stage did not vary significantly between treatments, indicating that increases in larval developmental time were responsible for the observed decrease in developmental rate. Mean time to mortality, a weighted average time of death, was not significantly affected by any of the treatments. In a 2 × 2 × 2 factorial analysis, all main effects (linear furanocoumarins.B. thuringiensis, UV radiation) reduced survival significantly, as did the three-way interaction. Thus, antagonistic interactions with psoralens that would reduce the effectiveness ofB. thuringiensis in the field were not observed. When pairs of main effects were nested within the two levels (presence and absence) of the third factor, several two-way interactions were found. Interestingly, the activity ofB. thuringiensis and the psoralens, individually or in combination, was enhanced by exposure to UV radiation. Implications of this research are discussed for both natural and agricultural ecosystems.Lepidoptera: Noctuidae.     

Tritrophic Effects of Xanthotoxin on the Polyembryonic Parasitoid <Emphasis Type="Italic">Copidosoma sosares</Emphasis> (Hymenoptera: Encyrtidae)

Plant chemistry can have deleterious effects on insect parasitoids, which include the reduction in body size, increased development time, and increased mortality. We examined the effects of xanthotoxin, a linear furanocoumarin, on the polyembryonic encyrtid wasp Copidosoma sosares, a specialist parasitoid that attacks the parsnip webworm, Depressaria pastinacella, itself a specialist on furanocoumarin-producing plants. Furanocoumarins, allelochemicals abundant in the Apiaceae and Rutaceae, are toxic to a wide range of herbivores. In this study, we reared parasitized webworms on artificial diets containing no xanthotoxin (control) or low or high concentrations of xanthotoxin. Clutch sizes of both male and female C. sosares broods were more than 20% smaller when they developed in hosts fed the diet containing high concentrations of xanthotoxin. Xanthotoxin concentration in the artificial diet had no effect on the development time of C. sosares, nor did it have an effect on the body size (length of hind tibia) of individual adult male and female C. sosares in single-sex broods. Webworms fed artificial diets containing low or high concentrations of xanthotoxin were not significantly smaller, and their development time was similar to that of webworms fed a xanthotoxin-free diet. Mortality of webworms was not affected by xanthotoxin in their artificial diet. Therefore, dietary xanthotoxin did not appear to affect C. sosares via impairment of host health. However, unmetabolized xanthotoxin was found in D. pastinacella hemolymph where C. sosares embryos develop. Hemolymph concentrations were fourfold greater in webworms fed the high-xanthotoxin-containing diet than in webworms fed the low-xanthotoxin-containing diet. We failed to detect any xanthotoxin metabolism by either C. sosares embryos or precocious larvae. Therefore, the observed tritrophic effects of xanthotoxin are likely to be due to the effects of xanthotoxin after direct contact in the hemolymph rather than to the effects of compromised host quality.     

Analysis,isolation and insecticidal activity of linear furanocoumarins and other coumarin derivatives fromPeucedanum (Apiaceae: Apioideae)

Peucedanum arenarium Waldst. & Kit.,P. austriacum (Jacq.) Koch,P. coriaceum Reichenb.,P. longifolium Waldst. & Kit,P. officinale L.,P. oreoselinum (L.) Moench,P. ostruthium L., andP. palustre (L.) Moench accumulate different structural types of coumarins including simple coumarins, linear furanocoumarins, linear dihydropyranocoumarins, angular dihydrofuranocoumarins and angular dihydropyranocoumarins. Linear furanocoumarins, known for various biological activities, include some well-known antifeedants, such as bergapten, isopimpinellin, and xanthotoxin. The aim of this investigation was to screen the diverse coumarins fromPeucedanum for insecticidal activity. LC was used to analyze and isolate coumarins for the bioassays. A growth inhibition bioassay with 17 derivatives, comprising all structural types fromPeucedanum, carried out withSpodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) as test organism, indicated the majority of the linear furanocoumarins and the angular dihydrofuranocoumarin athamantin as active compounds. Oxygenation of the prenyl residue of linear furanocoumarins decreased activity. Further formation of an ester with angelic acid even resulted in complete inactivity. Five active linear furanocoumarins, bergapten, isopimpinellin, xanthotoxin, isoimperatorin, and imperatorin, and two linear furanocoumarins with a substituted furan ring, peucedanin and 8-methoxypeucedanin, were compared in a dietary utilization bioassay. Relative growth rate (RGR) and relative consumption rate (RCR) divided the tested coumarins in three groups of similar activity. Isopimpinellin and peucedanin slightly decreased RGR and RCR of the treated larvae, and xanthotoxin, isoimperatorin, and 8-methoxypeucedanin heavily decreased RGR and RCR. Bergapten and imperatorin differed by the lowest RGR values and rather high RCR values. The effects caused by these two coumarins indicate specific postingestive toxicity. The results obtained in this study add to the reputation of coumarins to be an effective chemical defense, postulating that chemical diversity is a necessary trait for well-defended plants.     

Chemical ecology of the luna moth

The effects of food plant on larval performance and midgut detoxification enzymes were investigated in larvae of the luna moth,Actias luna. Neonate larvae were fed leaves of black cherry, cottonwood, quaking aspen, white willow, red oak, white oak, tulip tree, paper birch, black walnut, butternut, or shagbark hickory. First instar survival, larval duration, and pupal weights were monitored as indices of food quality. Midgut enzyme preparations from fifth instars were assayed for -glucosidase, quinone reductase, polysubstrate monooxygenase, esterase, and glutathione transferase activities. Larval survival on seven of the 11 plant species, including several recorded host plants, was extremely poor. Larvae performed well, and quite similarly, on birch, walnut, butternut, and hickory. Activities of all enzyme systems except -glucosidase were significantly influenced by larval host plant. Of the systems assayed, quinone reductase and glutathione transferase activities were especially high. Comparisons of these values with published values for other Lepidoptera support the hypothesis that these enzyme systems are involved in conferring tolerance to juglone and related quinones occurring in members of the plant family Juglandaceae. Results suggest that host plant utilization by luna is more specialized at the individual or population level than at the species level and that biochemical detoxification systems may play a role in such specialization.     

Cellulose digestion inMonochamus marmorator Kby. (Coleoptera: Cerambycidae): Role of acquired fungal enzymes

Larvae of the balsam fir sawyer,Monochamus marmorator Kby. (Coleoptera, Cerambycidae), contain midgut digestive enzymes active against hemicellulose and cellulose. Cellulases from larvae fed on balsam fir wood infected with the fungus,Trichoderma harzianum Rifai (Deuteromycetes, Moniliales, Moniliaceae), were found to be identical to those of the cellulase complex produced by this fungus when compared using chromatography, electrophoresis, and isofocusing. When larvae are maintained on a fungus-free diet, their midgut fluids lack cellulolytic activity, and they are unable to digest cellulose. Cellulolytic capacity can be restored by feeding the larvae wood permeated by fungi. We conclude that the enzymes which enableM. marmorator larvae to digest cellulose are not produced by the larvae. Instead, the larvae acquire the capacity to digest cellulose by ingesting active fungal cellulases while feeding in fungus-infected wood.     

Protective action of midgut catalase in lepidopteran larvae against oxidative plant defenses

Catalase activity was detected in the midgut tissues and regurgitate of several lepidopteran pests of the tomato plant. Greatest activity in the midgut was detected in larvalHelicoverpa zea, followed bySpodoptera exigua, Manduca sexta, andHeliothis virescens. We present evidence that catalase, in addition to removing toxic hydrogen peroxide, may inhibit the oxidation of plant phenolics mediated by plant peroxidases. Small amounts of larval regurgitate significantly inhibited foliar peroxidase activity via removal of hydrogen peroxide. Treatment of foliage with purified catalase nearly eliminated peroxidase activity and was superior as a larval food source compared to untreated foliage. Tomato foliar peroxidases oxidize an array of endogenous compounds including caffeic acid, chlorogenic acid, rutin, coumaric acid, cinnamic acid, and guaiacol. The oxidized forms of these compounds are potent alkylators of dietary and/or cellular nucleophiles (e.g., thiol and amino functions of proteins, peptides, and amines). When tomato foliar protein was pretreated with peroxidase and chlorogenic acid and incorporated in artificial diet, larval growth was reduced compared to larvae fed untreated protein. Thus, the diminution of peroxidase activity and removal of hydrogen peroxide by catalase may represent an important adaptation to leaf-feeding. The secretion of catalase in salivary fluid during insect feeding is also suggested to be a potential mechanism for reducing hydrogen peroxide formation as an elicitor of inducible plant defenses.     

Effect of Two Wheat Cultivars Differing in Hydroxamic Acid Concentration on Detoxification Metabolism in the AphidSitobion avenae

Hydroxamic acids (Hx) are wheat secondary metabolites conferring resistance for cereals against aphids. The activity of five enzymatic systems were evaluated in the aphid Sitobion avenae reared on the high-Hx wheat cultivar Chagual and the low-Hx wheat cultivar Huayún for 10 generations. Enzyme solutions were prepared from aphid homogenates and assayed for mixed function oxidases (including cytochrome P-450 monooxygenases and NADPH cytochrome c reductase), glutathione S-transferases, esterases, and catalase. Specific activities per aphid individual of cytochrome P-450 monooxygenases, NADPH cytochrome c reductase, glutathione S-transferases, and esterases were significantly increased in wheat cultivars relative to oat (only marginal increase of esterases in Chagual). Aphids fed on cv. Huayún showed an overall higher induction of enzymatic systems than those fed on cv. Chagual. Comparison of these results with reported effects of Hx on detoxifying enzymes in other insects, including aphids, support the hypothesis that these enzymatic pathways play an important role in the detoxification of toxic host-plant secondary metabolites.     

Effects of protein and juglone on gypsy moths: Growth performance and detoxification enzyme activity

The individual and interactive effects of dietary protein and juglone on larval performance and midgut detoxification enxymes were investigated for the gypsy moth,Lymantria dispar. The experimental design was a 2 × 3 factorial, with two levels of protein and three levels of juglone. We monitored survival/development rates from egg hatch to pupation and conducted fourth-instar feeding trials for determination of nutritional indices. Enzyme solutions were prepared from midguts of fifth instars and assayed for polysubstrate monooxygenase, esterase, quinone reductase, and glutathione transferase activities. Results showed that low protein levels prolonged development times, increased consumption rates, and reduced pupal weights. Juglone markedly reduced survival, growth, and consumption rates, increased development times, and reduced pupal weights. The interaction between protein and juglone influenced larval digestion efficiencies and female pupal weights. Polysubstrate monooxygenase activities were unaffected by diet, whereas esterase activities increased in response to both low dietary protein and presence of juglone. Low protein levels increased soluble quinone reductase activities but decreased glutathione transferase activities. Glutathione transferase activities were lowest in larvae fed low-protein, high-juglone diets and may have contributed to the especially poor performance of larvae on those diets. Quinone reductase and glutathione transferase are the systems of importance in detoxification of juglone, and moderate to low activities of these enzymes may explain why gypsy moths perform poorly on members of the Juglandaceae.     

Detoxication activity in the gypsy moth: Effects of host CO2 and NO 3 − availability

We investigated the effects of host species and resource (carbon dioxide, nitrate) availability on activity of detoxication enzymes in the gypsy moth,Lymantria dispar. Larvae were fed foliage from quaking aspen or sugar maple grown under ambient or elevated atmospheric CO₂, with low or high soil NO ₃ ^– availability. Enzyme solutions were prepared from larval midguts and assayed for activity of cytochrome P-450 monooxygenase, esterase, glutathione transferase, and carbonyl reductase enzymes. Activity of each enzyme system was influenced by larval host species, CO₂ or NO ₃ ^– availability, or an interaction of factors. Activity of all but glutathione transferases was highest in larvae reared on aspen. Elevated atmospheric CO₂ promoted all but transferase activity in larvae reared on aspen, but had little if any impact on enzyme activities of larvae reared on maple. High NO ₃ ^– availability enhanced activity of most enzyme systems in gypsy moths fed high CO₂ foliage, but the effect was less consistent for insects fed ambient CO₂ foliage. This research shows that gypsy moths respond biochemically not only to interspecific differences in host chemistry, but also to resource-mediated, intraspecific changes in host chemistry. Such responses are likely to be important for the dynamics of plantinsect interactions as they occur now and as they will be altered by global atmospheric changes in the future.     

A review of the antioxidant potential of medicinal plant species

Some researchers suggest that two-thirds of the world's plant species have medicinal value; in particular, many medicinal plants have great antioxidant potential. Antioxidants reduce the oxidative stress in cells and are therefore useful in the treatment of many human diseases, including cancer, cardiovascular diseases and inflammatory diseases. This paper reviews the antioxidant potential of extracts from the stems, roots, bark, leaves, fruits and seeds of several important medicinal species. Synthetic antioxidants such as butylated hydroxytoluene (BHT) and butylated hydroxylanisole (BHA) are currently used as food additives, and many plant species have similar antioxidant potentials as these synthetics. These species include Diospyros abyssinica, Pistacia lentiscus, Geranium sanguineum L., Sargentodoxa cuneata Rehd. Et Wils, Polyalthia cerasoides (Roxb.) Bedd, Crataeva nurvala Buch-Ham., Acacia auriculiformis A. Cunn, Teucrium polium L., Dracocephalum moldavica L., Urtica dioica L., Ficus microcarpa L. fil., Bidens pilosa Linn. Radiata, Leea indica, the Lamiaceae species, Uncaria tomentosa (Willd.) DC, Salvia officinalis L., Momordica Charantia L., Rheum ribes L., and Pelargonium endlicherianum. The literature reveals that these natural antioxidants represent a potentially side effect-free alternative to synthetic antioxidants in the food processing industry and for use in preventive medicine.