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.     

Lutein Sequestration and Furanocoumarin Metabolism in Parsnip Webworms Under Different Ultraviolet Light Regimes in the Montane West

Both biotic and abiotic selection pressures can contribute to geographic variation in allelochemical production in plants. We examined furanocoumarin production in western North American populations of Heracleum lanatum and Pastinaca sativa that, at different latitudes and altitudes, experience different ultraviolet (UV) light regimes. Total furanocoumarins and linear furanocoumarins of fruits were negatively correlated with UV irradiance, whereas amounts of angular furanocoumarins, which are generally less phototoxic, were not. Another factor potentially influencing furanocoumarin production is the presence of the parsnip webworm Depressaria pastinacella, (Lepidoptera: Oecophoridae), an herbivore that feeds on reproductive structures of both plant species. These insects sequester lutein from their host plants; this carotenoid acts to ameliorate furanocoumarin toxicity. Although the concentration of lutein in fruits did not vary with UV irradiance, lutein sequestration by sixth instars was positively correlated with UV irradiance. Webworm populations are variably infested with the polyembryonic webworm parasitoid Copidosoma sosares Walker (Hymenoptera: Encyrtidae). H. lanatum fruits from populations with webworms parasitized by C. sosares had lower concentrations of furanocoumarins, with the exception of sphondin, than fruits from plants infested with webworms free from parasitism. Lower levels of these furanocoumarins may reduce negative effects on the fitness of this parasitoid. In contrast with the variation in furanocoumarin content, the ability of webworms to metabolize furanocoumarins by cytochrome P450 did not differ significantly among populations from New Mexico to Alberta.     

Stratospheric ozone depletion and plantinsect interactions: Effects of UVB radiation on foliage quality ofCitrus jambhiri forTrichoplusia ni

Projected decreases in stratospheric ozone may result in increases in shortwave ultraviolet (UVB) irradiation at the earth's surface. Furanocoumarins, phototoxic compounds found inCitrus jambhiri foliage, increase in concentration when these plants are grown under enhanced UVB. Survivorship schedules ofTrichoplusia ni (Lepidoptera: Noctuidae) caterpillars reared on plants in the presence and absence of enhanced UVB regimes differ significantly; larvae develop more slowly in early life when reared on plants exposed to increased UVB. This same developmental pattern is observed whenT. ni larvae are reared on artificial diets amended with ecologically appropriate amounts of furanocoumarins. Thus, anthropogenically derived changes in stratospheric ozone and concomitant changes in UV light quality at the earth's surface may influence ecological interactions between insects and their host plants by altering secondary metabolism and hence foliage quality for herbivores.     

Defense of parsnip webworm against phototoxic furanocoumarins: Role of antioxidant enzymes

The parsnip webworm,Depressaria pastinacella (Lepidoptera: Oecophoridae), feeds on plants rich in furanocoumarins, phototoxic allomones. Final-instar larvae possess high levels of activities of antioxidant enzymes (Superoxide dismutase, catalase, glutathione reductase), which detoxify oxygen radicals generated from the furanocoumarins of their host plants. When added to an artificial diet, three linear furanocoumarins (xanthotoxin, bergapten, imperatorin) do not increase levels of the antioxidant enzymes. However, on diets containing both xanthotoxin and piperonyl butoxide, a cytochrome P-450 inhibitor, food utilization indices of the insect are reduced and superoxide dismutase activity is enhanced. These data suggest that cytochrome P-450s act as a primary detoxification system of ingested furanocoumarin, and antioxidant enzymes as a backup system to detoxify oxygen radicals generated by unmetabolized furanocoumarins.     

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.     

Decreased sensitivity of mixed-function oxidases frompapilio polyxenes to inhibitors in host plants

Caterpillars ofPapilio polyxenes, the black swallowtail, feed on umbellifers that contain both toxic furanocoumarins and methylenedioxyphenyl compounds such as myristicin and safrole. These phytosynergists enhance the toxicity of furanocoumarins by inhibiting mixed-function oxidases (MFOs), the detoxification enzymes responsible for metabolizing furanocoumarins. In model substrate assays, MFOs fromP. polyxenes are twice as active as MFOs fromHeliothis zea, a generalist herbivore not adapted to feeding on either furanocoumarins or furanocoumarin/phytosynergist combinations.P. polyxenes MFOs are 10 and 46 times less sensitive to inhibition by myristicin and safrole, respectively, thanH. zea MFOs and eight times less sensitive to inhibition by safrole than MFOs fromPapilio troilus, a closely related species that does not encounter furanocoumarin/phytosynergist combinations in its diet. Higher MFO activity and decreased sensitivity to MFO inhibitors are important adaptations that allow black swallowtail caterpillars to feed on many umbelliferous plants.     

Dietary and developmental influences on induced detoxification in an oligophage

Many plant secondary compounds induce detoxification activity in herbivorous insects. Although inducibility may be advantageous as a means of reducing costs associated with maintenance of metabolism, another benefit of inducibility is that it may allow insects to tailor their detoxification profiles to multiple substrate toxins in their diets. The parsnip webworm, Depressaria pastinacella, must contend with many types of furanocoumarins, toxins present in abundance in all of its host plants. Previous studies have documented that cytochrome P-450s are responsible for metabolism of furanocoumarins in this species and that this overall activity is inducible. In this study, we examined the effects of ingestion of single furanocoumarins on metabolism of multiple furanocoumarins and the ability of webworms to adjust their metabolism profiles to match artificial diets with furanocoumarin content differing qualitatively and quantitatively from the average content found in their principal host. That detoxification rates of newly molted sixth instars prior to feeding did not differ from those of actively feeding fifth or sixth instars suggests that constitutive activities of furanocoumarin-metabolizing enzymes are maintained in the absence of substrates. All of the induction assays in this study were performed with ultimate instars. Each of the furanocoumarins assayed was found to induce metabolism of five different furanocoumarin substrates; however, the induction profile was independent of the inducing agent. Consistent with this finding, webworms were incapable of matching their detoxification profiles to diets with different furanocoumarin compositions. Thus, the profile of detoxification within individuals of this species appears to be genetically fixed, although there is considerable variation in profiles among individuals.     

Artificial defloration and furanocoumarin induction inPastinaca sativa (Umbelliferae)

Damage simulating herbivory was tested as an inducer of furanocoumarins in the floral parts of the wild parsnip,Pastinaca sativa (Umbelliferae). In one experiment, primary umbels ofP. sativa were partially deflorated over the course of nine days, and higher-order umbels as well as the remaining primary umbel floral parts were sampled. Total furanocoumarin concentration was not significantly affected by defloration, but one furanocoumarin, isopimpinellin, increased in one floral stage of the secondary umbel in damaged plants. In a second experiment, primary umbels were completely deflorated and the higher-order umbels allowed to set seed. No significant effect of defloration on furanocoumarin content was found in the seeds of the higher-order umbels.     

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.     

Laboratory colonization has not reduced constitutive or induced polysubstrate monooxygenase activity in velvetbean caterpillars

We evaluated whether velvetbean caterpillars (Anticarsia gemmatalis) from a laboratory colony had reduced constitutive (basal) and/or induced activities of their polysubstrate monooxygenase (PSMO) detoxification enzyme system as a result of long-term rearing (> 100 generations) on artificial diet without introduction of field-collected individuals. Larvae from the laboratory colony and those from a recently collected field strain were fed either a standard artificial diet (control), one containing the inducing allelochemical, flavone, or foliage ofIndigofera hirsuta (a host plant of this species), and their midgut PSMO activity was assessed by measuring the in vitro rate of aldrin epoxidation. Compared with the field-strain larvae, caterpillars from the laboratory colony had 1.9-fold greater constitutive activity (standard artificial diet) and 2.3-fold greater induced activity (flavone-treated diet). In addition, the magnitude of induction was somewhat greater for the laboratory-colony larvae (induced activity was 2.0-fold greater than constitutive activity) compared with those from the field-strain (1.6-fold). In contrast, no difference in strain activity was found when larvae were fedI. hirsuta foliage. The lower PSMO activity of the field-strain larvae when fed artificial diet may have been caused by their reduced feeding and growth performance compared with laboratory-colony larvae, and it may explain their greater sensitivity to allelochemicals incorporated in the artificial diet, as we found previously. The results of this study indicate that long-term rearing of theA. gemmatalis laboratory colony on artificial diet, without the introduction of field individuals, apparently has not selected for low constitutive activity or decreased inducibility of PSMO, and thus these larvae provide a suitable model for studying xenobiotic detoxication. In addition, they suggest that using an artificial diet to evaluate resistance to pesticides or other xenobiotics in fieldcollected insects, as is frequently done, may underestimate the level of resistance if the diet, through various causes, reduces the activity of detoxification enzymes contributing to the resistance.     

Food experience on the predatory behavior of the ant Myrmica rubra towards a specialist moth,Acrolepiopsis assectella

Entomophagous insects are often repelled by the secondary compounds of the plants eaten by their prey. These compounds, therefore, take on a defensive role for the phytophagous species that sequester them. Given that numerous entomophagous species are capable of learning, the effects on the foraging behavior of a repeated experience were investigated in the predatory ant Myrmica rubra. The sulfur amino acids methyl-cysteine sulfoxide (MCSO) and propyl-cysteine sulfoxide (PCSO) produced by Allium plants were identified in caterpillars of the leek moth Acrolepiopsis assectella. Three behavioral studies were carried out, with or without prior familiarization with caterpillars reared either on leek or on an artificial diet containing no Allium compounds. In choice tests with the two types of caterpillars, unfamiliarized ants displayed a preference for caterpillars reared on the artificial diet, but this preference disappeared or was reversed in both young and old ants after familiarization.     

Host plant utilization and iridoid glycoside sequestration byEuphydryas anicia (Lepidoptera: Nymphalidae)

The iridoid glycoside content of individual adultEuphydryas anicia butterflies from two Colorado populations was quantitatively determined. At one site (Red Hill), larval host plants wereCastilleja integra andBesseya plantaginea, while at the other site (Cumberland Pass) a single host plant,B. alpina, was used. At Red Hill, macfadienoside and catalpol were sequestered, while at Cumberland Pass, catalpol and aucubin were sequestered. Artificial diet studies showed that larvae hydrolyzed a major iridoid ofB. plantaginea, 6-isovanilIylcatalpol, to catalpol (which was sequestered) and isovanillic acid (which was excreted). Large year-to-year and individual variation in butterfly iridoid content was established as was a female-male difference in macfadienoside vs. catalpol content. Larval host plant distributions and numbers were determined at Red Hill for two years and compared with changes in butterfly populations and sequestered iridoids.     

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.     

Psoralens in senescing leaves ofRuta graveolens

Concentrations of three furanocoumarins, psoralen, xanthotoxin, and bergapten, were measured on the surface and within mature whole leaves of two groups ofRuta graveolens L. late autumn plants, 2 and 6 years old, which contained green, yellow, and dry yellow leaves. Upper green leaves contained higher concentrations of these coumarins than lower green leaves, green leaves contained several times as much as yellow leaves, and dry leaves contained even smaller amounts than yellow ones. The dry yellow leaves contained only a very small percentage of furanocoumarins on the surface, suggesting that extrusion to the surface of yellow leaves was slower or had stopped, while loss from the surface continued. The loss of psoralen was the most dramatic in and on the dry leaves. Bergapten's ratio to the other cournarins increased during senescence. Xanthotoxin was always the predominant furanocoumarin in this species.     

Reduction in diet breadth results in sequestration of plant chemicals and increases efficacy of chemical defense in a generalist grasshopper

The lubber grasshopper,Romalea guttata, is a generalist feeding on a broad diet of many herbaceous plant species and has a metathoracic defensive secretion normally containing phenolics and quinones synthesized by the insect. When insects were reared on a restricted diet of wild onion, they sequestered sulfur volatiles from the plant into their defensive secretions. These compounds were not detected by gas chromatography-mass spectroscopy in secretions of insects on an artificial diet or a natural, generalist diet of 26 plants that included wild onion as a component, nor were they present in secretions from field-collected insects. Defensive secretions of insects reared on wild onion were significantly more deterrent, by as much as an order of magnitude, to two species of ant predators than secretions from insects on either of the other two diets, despite a reduction in the concentration of autogenous defensive chemicals in secretions of insects on the onion diet. Sequestration of plant chemicals that increased defensive efficacy occurred when diet breadth was reduced. We suggest that this occurs because under conditions of specialization, plant secondary metabolites are more likely to be ingested and bioaccumulated in sufficient concentrations to have biological activity against predators. What we define as casual bioaccumulation of bioactive plant chemicals following dietary specialization may lead to evolution of sequestered defense syndromes in insects, and this process may not necessarily require specific adaptation to or coevolution with a toxic host plant.     

Cucurbitacins

Four species of diabroticites with different host specificities are shown for the first time to sequester cucurbitacins. While all beetles fed on an artificial diet (no cucurbitacins) were readily consumed by Chinese praying mantids, a significant proportion of adultDiabrotica balteata (72%),D. undecimpunctata howardi (46%), andD. virgifera virgifera (24%) fed on squash fruit containing cucurbitacins B and D were rejected. Moreover, even when adults did not feed on cucurbitacins, 21–24% ofAcalymma vittatum were rejected by the mantids which is consistent with larval sequestration of cucurbitacins. The mantids failed to learn to avoid any of the beetle species despite adverse effects associated with ingestion, i.e., uncoordination, regurgitation, etc. A cucurbitacin D metabolite accumulated and was sequestered for extended periods of time in the hemolymph of all four species. In addition, female beetles that had ingested cucurbitacins laid eggs containing substantial amounts of cucurbitacins.     

Flower <Emphasis Type="Italic">vs.</Emphasis> Leaf Feeding by <Emphasis Type="Italic">Pieris brassicae</Emphasis>: Glucosinolate-Rich Flower Tissues are Preferred and Sustain Higher Growth Rate

Interactions between butterflies and caterpillars in the genus Pieris and plants in the family Brassicaceae are among the best explored in the field of insect–plant biology. However, we report here for the first time that Pieris brassicae, commonly assumed to be a typical folivore, actually prefers to feed on flowers of three Brassica nigra genotypes rather than on their leaves. First- and second-instar caterpillars were observed to feed primarily on leaves, whereas late second and early third instars migrated via the small leaves of the flower branches to the flower buds and flowers. Once flower feeding began, no further leaf feeding was observed. We investigated growth rates of caterpillars having access exclusively to either leaves of flowering plants or flowers. In addition, we analyzed glucosinolate concentrations in leaves and flowers. Late-second- and early-third-instar P. brassicae caterpillars moved upward into the inflorescences of B. nigra and fed on buds and flowers until the end of the final (fifth) instar, after which they entered into the wandering stage, leaving the plant in search of a pupation site. Flower feeding sustained a significantly higher growth rate than leaf feeding. Flowers contained levels of glucosinolates up to five times higher than those of leaves. Five glucosinolates were identified: the aliphatic sinigrin, the aromatic phenyethylglucosinolate, and three indole glucosinolates: glucobrassicin, 4-methoxyglucobrassicin, and 4-hydroxyglucobrassicin. Tissue type and genotype were the most important factors affecting levels of identified glucosinolates. Sinigrin was by far the most abundant compound in all three genotypes. Sinigrin, 4-hydroxyglucobrassicin, and phenylethylglucosinolate were present at significantly higher levels in flowers than in leaves. In response to caterpillar feeding, sinigrin levels in both leaves and flowers were significantly higher than in undamaged plants, whereas 4-hydroxyglucobrassicin leaf levels were lower. Our results show that feeding on flower tissues, containing higher concentrations of glucosinolates, provides P. brassicae with a nutritional benefit in terms of higher growth rate. This preference appears to be in contrast to published negative effects of volatile glucosinolate breakdown products on the closely related Pieris rapae.     

Mechanisms of behavioral alterations of parasitoids reared in artificial systems

A high quality of mass reared parasitoids is required for successful biological control of pest insects. Although the phenomenon of behavioral deterioration of parasitoids due to rearing in artificial conditions is well known, its significance is often underestimated, and the underlying mechanisms are poorly investigated. We quantified behavioral alterations of parasitoids reared in an artificial system vs. a natural system and elucidated some of the mechanisms involved. The model systems consisted of apple fruits (natural system) or an artificial diet devoid of apple (artificial system), the herbivore Cydia pomonella, and its larval parasitoid Hyssopus pallidus, a candidate biological control agent. Two parasitoid strains, one reared for 30 generations in the natural system and one in the artificial system, were compared by using the females' ability to respond to frass from codling moth caterpillars fed on apple fruits (apple-frass). The searching response of parasitoids reared in the artificial system compared to those reared in the natural system was reduced by an average of 53.2%. Gas chromatography–mass spectrometry (GC-MS) analyses of the two types of caterpillars' food and of the two corresponding types of frass showed that 15 compounds were present only in apple fruits and apple-frass, three compounds only in artificial diet and artificial-diet-frass, while four compounds were present in both frass types but not in the food sources. This suggests the presence of a food-derived and a host-derived component in the frass. Results from both bioassays and chemical analyses indicate that the kairomonal activity of the frass is due to both apple fruit and host components. The reduced response of parasitoids reared in artificial conditions might, therefore, be due to a lack of recognition of the apple fruit component. In a further experiment, the two parasitoid strains were reared in the opposite system for one generation. While the response to the host frass was significantly reduced in parasitoids that emerged from the artificial system, it was fully restored in parasitoids that emerged from the natural system. This indicates that the behavioral alteration was related to a learning process during ontogenesis rather than to a selection exhibited over generations.     

Fate of iridoid glycosides in different life stages of the Buckeye,Junonia coenia (Lepidoptera: Nymphalidae)

The buckeye butterfly,Junonia coenia (Lepidoptera: Nymphalidae), specializes on plants that contain iridoid glycosides. To determine the fate of these compounds in larvae, pupae, and adults of this species, we reared larvae on artificial diets with and without iridoid glycosides, and on leaves of a host plant,Plantago lanceolata (Plantaginaceae). Quantification by gas chromatography showed that newly molted third-, fourth-, and fifth-instar larvae reared on leaves ofP. lanceolata contained means of 5.13, 2.88, and 6.83% dry weight iridoid glycoside. In contrast, the mean iridoid glycoside concentration of actively feeding fifth-instar larvae was 0.28% dry weight, that of pupae was 0.19% dry weight iridoids, and adults contained no detectable iridoids. Feeding experiments suggested that this reduction in actively feeding larvae was due to the metabolism of iridoid glycosides.P. lanceolata leaves in these experiments contained a mean of 1.00% dry weight iridoid glycoside, with a 2:1 ratio of aucubin to catalpol. Calculation of iridoid consumption and utilization indices showed that larvae fed artificial diets consumed, digested, and sequestered aucubin and catalpol in similar ways. When these indices were calculated for larvae fed leaves ofP. lanceolata, catalpol was sequestered twice as efficiently as aucubin.