Selection for chemical trait remixing in an invasive weed after reassociation with a coevolved specialist.

The interaction between Depressaria pastinacella (parsnip webworm) and wild parsnip (Pastinaca sativa), in its native Europe and in its longstanding nonindigenous range in the midwestern United States, is characterized by chemical phenotype matching, ostensibly mediated by reciprocal selective responses. The first appearance of D. pastinacella on P. sativa in New Zealand in 2004 provided an opportunity to quantify selective impacts of a coevolved herbivore and calibrate rates of phytochemical response in its host plant. Webworms in 2006 reduced seed production up to 75% in New Zealand populations, and in 2007 infestations increased in severity in all populations except one. Most New Zealand populations fall into a furanocoumarin phenotype cluster distinct from European and U.S. phenotypes, although one heavily attacked population clusters with two U.S. populations and one European population long associated with webworms. Multivariate selection analysis substituting realized fitness (with webworms present) for potential fitness (absent webworms) as the dependent variable revealed that reassociation with a coevolved specialist in a nonindigenous area profoundly altered the selection regime, favoring trait remixing and rapid chemical changes in parsnip populations, as predicted by the geographic mosaic theory. That uninfested populations of New Zealand parsnips contain higher amounts of octyl acetate, a floral volatile used by webworms for orientation, suggests that plants that escape from specialized enemies may also experience selection to increase kairomones, as well as to reduce allomones.

Remarkable substrate-specificity of CYP6AB3 in Depressaria pastinacella, a highly specialized caterpillar.

The parsnip webworm, Depressaria pastinacella, a specialist on two genera in Apiaceae, feeds exclusively on the furanocoumarin-containing reproductive structures of its host plants. This caterpillar relies principally on cytochrome P450-mediated detoxification for coping with the high concentrations of furanocoumarins in its diet. A cDNA encoding the furanocoumarin-inducible P450 CYP6AB3 from this species was coexpressed with house-fly NADPH P450 reductase in baculovirus-infected Sf9 cells and tested for binding and metabolism of the six furanocoumarins typically encountered in host plant tissues. Only imperatorin and bergapten bind in close proximity to the catalytic haem and only imperatorin is metabolized (V(max) and K(m) of 2.412 pmol/min per pmol P450 and 94.28 microm, respectively). Purification of the imperatorin metabolite by normal phase HPLC and characterization of its structure by MS-MS analysis indicate that CYP6AB3 initially epoxidizes the carbon-carbon pi-bond on the isoprenyl side chain on imperatorin. An improved molecular model for the CYP6AB3 protein based on this biochemical characterization and the recently defined mammalian CYP3A4 crystal structure provides insight into the remarkable substrate specificity of this protein.

Characterization and evolution of furanocoumarin-inducible cytochrome P450s in the parsnip webworm, Depressaria pastinacella.

Depressaria pastinacella, the parsnip webworm, a specialist on two genera in the Apiaceae, routinely consumes plant tissues high in furanocoumarin content and is capable of rapid cytochrome P450-mediated detoxification of these compounds. In this study, four cDNAs were cloned from the larval midgut of this insect: two full-length CYP6AB3 and CYP6AE1 cDNAs are closely related to members of the furanocoumarin-metabolizing CYP6B subfamily and two partial CYP9A6 and CYP9A7 cDNAs are related to members of the CYP9A subfamily that have also been linked to the detoxification of xenobiotics. At least one of these P450s (CYP6AB3) is inducible by dietary furanocoumarins, indicating its potential involvement in furanocoumarin metabolism. A homology model of CYP6AB3 was constructed and compared to models of CYP6B1 from the specialist species, Papilio polyxenes, and CYP6B4 from the generalist species, P. glaucus. Structural superpositioning of these models has revealed very high spatial similarity of elements, including the B helix, B'-C loop, I helix and C-terminal domain, within the catalytic sites of these proteins. Most importantly, key amino acid residues that can potentially come into contact with furanocoumarin substrates display conservation in their spatial positioning and side chain polarities. Three of these residues, Val103, Leu113 and Phe118 (numbered according to CYP6AB3), are conserved in all three of these proteins, further implicating CYP6AB3 in furanocoumarin metabolism by parsnip webworms. Characterization of these P450 cDNAs will allow for functional analyses aimed at elucidating the molecular mechanisms underlying the coevolutionary interactions between this herbivore and its principal host plant.

Phenotype matching in wild parsnip and parsnip webworms: causes and consequences.

According to the geographic mosaic theory of coevolution, selection intensity in interactions varies across a landscape, forming a selection mosaic; interaction traits match at coevolutionary hotspots where selection is reciprocal and mismatch at coldspots where reciprocity is not a factor. Chemical traits play an important role in the interaction between wild parsnip (Pastinaca sativa) and the parsnip webworm (Depressaria pastinacella). Furanocoumarins, produced as plant defenses, are detoxified by the webworms by cytochrome P450 monooxygenases; significant additive genetic variation exists for both furanocoumarin production in the plant and detoxification in the insect, making these traits available for selection. To test the hypothesis that differences in selection intensity affect the distribution of coevolutionary hotspots and coldspots in this interaction, we examined 20 populations of webworms and wild parsnips in Illinois and Wisconsin that varied in size, extent of infestation, proximity to woods (and potential vertebrate predators), and proximity to a chemically distinct alternate host plant, Heracleum lanatum (cow parsnip). Twelve of 20 populations displayed phenotype matching between plant defense and insect detoxification profiles. Of the eight mismatched populations, a logistic regression model related matching probability to two predictors: the presence of the alternate host and average content of xanthotoxin (one of the five furanocoumarins produced by P. sativa). The odds of mismatching were significantly increased by the presence of the alternate host (odds ratio = 15.4) and by increased xanthotoxin content (odds ratio = 6.053). Parsnips growing near cow parsnip displayed chemical phenotypes that were chemically intermediate between cow parsnip and parsnips growing in isolation. Rapid phenotype matching in this system is likely due in part to differential mortality every season; larvae transferred to a plant 30 m or more from the plant on which they developed tended to experience increased mortality over larvae transferred to another umbel on the same plant on which they had developed, and plant populations that mismatched in 2001 displayed a change in chemical phenotype distribution from the previous year. Trait mixing through gene flow is also a likely factor in determining mismatch frequency. Populations from which webworms were eradicated the previous year were all recolonized; in three of seven of these populations, infestation rates exceeded 90%. Our findings, consistent with the geographic mosaic theory, suggest that the presence of a chemically distinct alternate host plant can affect selection intensity in such a way as to reduce the likelihood of reciprocity in the coevolutionary interaction between wild parsnip and the parsnip webworm.

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.

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.

Impact of folivory on photosynthesis is greater than the sum of its holes.

The effects of herbivores on plant production and fitness may not relate directly to the quantity of biomass removed because folivory may alter photosynthetic rates at a considerable distance from the damaged tissue [Welter, S. C. (1989) in Insect-Plant Interactions, ed. Bernays, E. A. (CRC, Boca Raton), pp. 135-151.]. An impediment to understanding the effects of leaf damage on photosynthesis has been an inability to map photosynthetic function within a single leaf. We developed an instrument for imaging chlorophyll fluorescence and used it to map the effects of caterpillar feeding on whole-leaf photosynthesis in wild parsnip. The adverse effects of caterpillar feeding on photosynthesis were found to extend well beyond the areas of the leaflet in which caterpillars removed tissue. These "indirectly" affected areas remained impaired for at least 3 days after the caterpillars were removed and were six times as large as the area directly damaged by the caterpillars. Although photosynthesis in indirectly affected areas was reduced and not eliminated, these areas accounted for three times as much of the overall reduction in photosynthesis as the area removed by the caterpillars. The size of the indirect effects was positively correlated with defense-related synthesis of furanocoumarins, suggesting that costs of chemical defense may be one factor that accounts for the indirect effects of herbivory on plants.

Developmental variation in cytochrome P450 expression in Papilio polyxenes in response to xanthotoxin, a hostplant allelochemical.

Although developmental variation in activity and inducibility is typical of cytochrome P450 monooxygenases (P450s) in insects, the adaptive significance of such variation is often unclear, in part because the natural function of insect P450s is rarely known. In this study, we examined developmental variation in expression of CYP6B1 and CYP6B3 in Papilio polyxenes, the black swallowtail. Enzymes encoded by these genes have been implicated in the metabolism of xanthotoxin, a furanocoumarin characteristic of the apiaceous hostplants of P. polyxenes. In each life stage-egg, five larval instars, pupa, and adult-we examined individuals exposed to foliage with and without supplemental xanthotoxin. For each stage, we conducted enzyme assays to estimate xanthotoxin metabolism, Northern analysis to detect constitutive and induced mRNA levels, and RT-PCR amplification and Southern analysis to differentiate among P450 genes expressed. Inducible xanthotoxin metabolism, previously reported in fifth instars, was observed in four of five larval stages but was absent or undetectable in all stages that do not feed on foliage; the highest levels of activity were in early larval instars. The same pattern was observed in both Northern and RT-PCR gel blot analyses. In inducible larval stages, inducibility of CYP6B1 transcripts by xanthotoxin was greater than the inducibility of CYP6B3 transcripts. These findings support earlier suggestions that these two P450s contribute to xanthotoxin metabolism in this species and that expression of these P450 genes is regulated in an adaptive fashion with respect to probability of exposure to hostplant toxins over the course of development.

Effects of phytic acid and xanthotoxin on growth and detoxification in caterpillars.

Phytic acid is abundant in the fruits and seeds of many plants and is found in foliage to a lesser extent. Among its several properties, phytic acid is a potent chelator of essential minerals and proteins; thus, the possibility exists that heme-based enzymes such as cytochrome P450 monooxygenases in herbivores are detrimentally affected by phytic acid via chelation of dietary iron. Mortality, growth performance, and P450-mediated metabolism of xanthotoxin, a plant allelochemical, were examined in the presence of phytic acid in three lepidopteran species: a polyphagous seed-feeding species (Heliothis virescens), a polyphagous foliage-feeding species (Trichoplusia ni), and a species oligophagous on immature reproductive structures of two genera of Apiaceae (Depressaria pastinacella). While first instar H. virescens experienced no increase in mortality after 120 hours on a diet containing 1% phytic acid compared to a control diet, both T. ni and D. pastinacella experienced virtually complete mortality over the same time period. Ultimate instars of all three species experienced reductions in relative growth rates (RGR) and relative consumption rates (RCR) in the presence of phytic acid, although the only species to experience reduced digestive efficiency (ECI) was H. virescens. Cytochrome P450-mediated metabolism of xanthotoxin was reduced 60% in the presence of phytic acid in D. pastinacella, although metabolism remained unaffected in the two noctuids. These studies suggest a defensive function of phytic acid in addition to its primary functions of phosphorus storage, energy storage, and cell wall precursor source.

Effects of exposure to event 176 Bacillus thuringiensis corn pollen on monarch and black swallowtail caterpillars under field conditions.

The widespread planting of corn genetically modified to produce Bacillus thuringiensis endotoxin has led to speculation that pollen from these fields might adversely affect nearby nontarget lepidopterans. A previous study of Bt corn engineered with Monsanto event 810 failed to detect an effect of pollen exposure on the black swallowtail, Papilio polyxenes, in either the field or the laboratory. Here, we report results of a field study investigating the impact of exposure to pollen from a Bt corn hybrid containing Novartis event 176 on two species of Lepidoptera, black swallowtails and monarch butterflies, Danaus plexippus. Nearly half of the 600 monarch larvae died within the first 24 h; this and subsequent mortality was not associated with proximity to Bt corn and may have been due in part to predation. Survivorship of black swallowtails was much higher than that of the monarchs and was also independent of proximity to the transgenic corn. However, despite five rainfall events that removed much of the pollen from the leaves of their host plants during the experiment, we observed a significant reduction in growth rates of black swallowtail larvae that was likely caused by pollen exposure. These results suggest that Bt corn incorporating event 176 can have adverse sublethal effects on black swallowtails in the field and underscore the importance of event selection in reducing environmental impacts of transgenic plants.

Expression of CYP6B1 and CYP6B3 cytochrome P450 monooxygenases and furanocoumarin metabolism in different tissues of Papilio polyxenes (Lepidoptera: Papilionidae).

The CYP6B1 and CYP6B3 cytochrome P450 monooxygenases in the midgut of the black swallowtail participate in the metabolism of toxic furanocoumarins present in its host plants. In this study, biochemical analyses indicate that the fat body metabolizes significant amounts of the linear furanocoumarins bergapten and xanthotoxin after larvae feed on xanthotoxin. Northern analyses of the combined CYP6B1/3 transcript expression patterns indicate that transcripts in this P450 subfamily are induced in the midgut and fat body by xanthotoxin. Semi-quantitative RT-PCR analyses of individual CYP6B1/CYP6B3 mRNAs indicate that CYP6B1 transcripts are induced by xanthotoxin in all tissues examined and that CYP6B3 transcripts are induced in the fat body only. These results indicate that the fat body participates in the P450-mediated metabolism of excess furanocoumarins unmetabolized by the midgut. Although transcripts of both genes were detected and CYP6B1 transcripts were induced by xanthotoxin in the integument, furanocoumarin metabolism was not detected. Comparison of these P450 promoters with the promoters of alcohol dehydrogenase genes expressed in the fat bodies of several Drosophila species suggest that the xanthotoxin inducibilities of these P450 genes in fat bodies are regulated by elements other than those modulating expression of Adh genes.

Honeys from different floral sources as inhibitors of enzymatic browning in fruit and vegetable homogenates.

Honeys from different floral sources were evaluated for their antioxidant content and for their ability to inhibit enzymatic browning in fruits and vegetables. Antioxidant contents of honeys vary widely from different floral sources, as do their abilities to protect against enzymatic browning. Polyphenol oxidase (PPO) activity was reduced over a range of approximately 2-45% in fruit and vegetable homogenates, corresponding to a reduction in browning index by 2.5-12 units. Soy honey was particularly effective when compared to clover honey, which had a similar antioxidant content. When compared to commercial inhibitors of browning, honeys were less effective; however, in combination they added to the effectiveness of metabisulfite and ascorbic acid. Honey has great potential to be used as a natural source of antioxidants to reduce the negative effects of PPO browning in fruit and vegetable processing.

Absence of toxicity of Bacillus thuringiensis pollen to black swallowtails under field conditions.

A single laboratory study on monarch butterflies has prompted widespread concern that corn pollen, engineered to express Bacillus thuringiensis (Bt) endotoxin, might travel beyond corn fields and cause mortality in nontarget lepidopterans. Among the lepidopterans at high potential risk from this technology is the black swallowtail butterfly, Papilio polyxenes, whose host plants in the midwestern U. S. are located principally in narrow strips between roads and crop fields. A field study was performed to assess whether mortality of early instar black swallowtails was associated either with proximity to a field of Bt corn or by levels of Bt pollen deposition on host plants. Potted host plants were infested with first instar black swallowtails and placed at intervals from the edge of a field of Bt corn (Pioneer 34R07 containing Monsanto event 810) at the beginning of anthesis. We confirmed by ELISA that pollen from these plants contained Cry1Ab endotoxin (2.125 +/- 0.289 ng/g). Although many of the larvae died during the 7 days that the experiments were run, there was no relationship between mortality and proximity to the field or pollen deposition on host plants. Moreover, pollen from these same plants failed to cause mortality in the laboratory at the highest pollen dose tested (10,000 grains/cm(2)), a level that far exceeded the highest pollen density observed in the field (200 grains/cm(2)). We conclude that Bt pollen of the variety tested is unlikely to affect wild populations of black swallowtails. Thus, our results suggest that at least some potential nontarget effects of the use of transgenic plants may be manageable.

Heritability estimates for octyl acetate and octyl butyrate in the mature fruit of the wild parsnip.

The aliphatic esters octyl acetate and octyl butyrate occur as major components of essential oils in the vittae, or oil tubes, of the wild parsnip (Pastinaca sativa). We determined phenotypic variation and narrow-sense heritabilities of these octyl esters in wild parsnip fruits from 30 maternal families. The mean octyl acetate content was 1.56 microg/mg dry fruit (0.08-5.51 microg/mg dry fruit) and the mean octyl butyrate content was 4.28 microg/mg dry fruit (1.28-14.22 microg/ mg dry fruit). Narrow-sense heritabilities for each ester's content were calculated by analysis of half-sib families (HS) and parent-offspring regression (OP). Heritabilities were 0.389 (HS) and 0.654 (OP) for octyl acetate and 0.670 (HS) and 0.626 (OP) for octyl butyrate. The amounts of the esters were phenotypically correlated with each other and with the linear furanocoumarins bergapten and xanthotoxin, phototoxic compounds that co-occur in the vittae with the esters. Ester amounts were not genetically correlated, indicating that these compounds could respond independently to selection pressures. These octyl esters may serve as carrier solvents that enhance penetration of these furanocoumarins into herbivore integuments and gut walls.

Cross-resistance to alpha-cypermethrin after xanthotoxin ingestion in Helicoverpa zea (Lepidoptera: Noctuidae).

Cytochrome P450 monooxygenases (P450) are membrane-bound hemoproteins that play important roles in conferring protection against both naturally occurring phytochemicals and synthetic organic insecticides. Despite the potential for common modes of detoxification, cross-resistance between phytochemicals and synthetic organic insecticides has rarely been documented. In this study, we examined the responses of a susceptible strain of corn earworm, Helicoverpa zea (Boddie), a polyphagous noctuid, to exposure by an allelochemical infrequently encountered in its host plants and by an insecticide widely used for control purposes. Within a single generation, survivors of xanthotoxin exposure displayed higher levels of tolerance to alpha-cypermethrin than did unexposed control larvae. The F1 offspring of xanthotoxin-exposed survivors also displayed higher alpha-cypermethrin tolerance than did offspring of unexposed control larvae, suggesting that increased alpha-cypermethrin tolerance after xanthotoxin exposure represents, at least in part, heritable resistance. Administration of piperonyl butoxide, a P450 synergist, demonstrated that resistance to both xanthotoxin and alpha-cypermethrin is P450-mediated. Alpha-cypermethrin-exposed survivors, however, failed to show superior growth on xanthotoxin diets. Assays with control larvae, larvae induced by both xanthotoxin and alpha-cypermethrin, and survivors of LD50 doses of both compounds indicated that H. zea midgut P450s are capable of metabolizing both xanthotoxin and alpha-cypermethrin. Metabolism of each compound is significantly inhibited by the presence of the other compound, suggesting that at least one form of P450 in H. zea midguts degrades both compounds and may constitute the biochemical basis for possible cross-resistance. Compared with control larvae, xanthotoxin- and alpha-cypermethrin-induced larvae displayed 2- to 4-fold higher P450-mediated metabolism of both compounds. However, xanthotoxin- and alpha-cypermethrin-exposed survivors exhibited much higher (2.5- to 11-fold) metabolism of both compounds than did the induced larvae. The metabolism results, like the bioassay results, are consistent with the interpretation that increased alpha-cypermethrin tolerance after xanthotoxin exposure is attributable mainly to heritable resistance.

Chemical phenotype matching between a plant and its insect herbivore.

Two potential outcomes of a coevolutionary interaction are an escalating arms race and stable cycling. The general expectation has been that arms races predominate in cases of polygenic inheritance of resistance traits and permanent cycling predominates in cases in which resistance is controlled by major genes. In the interaction between Depressaria pastinacella, the parsnip webworm, and Pastinaca sativa, the wild parsnip, traits for plant resistance to insect herbivory (production of defensive furanocoumarins) as well as traits for herbivore "virulence" (ability to metabolize furanocoumarins) are characterized by continuous heritable variation. Furanocoumarin production in plants and rates of metabolism in insects were compared among four midwestern populations; these traits then were classified into four clusters describing multitrait phenotypes occurring in all or most of the populations. When the frequency of plant phenotypes belonging to each of the clusters is compared with the frequency of the insect phenotypes in each of the clusters across populations, a remarkable degree of frequency matching is revealed in three of the populations. That frequencies of phenotypes vary among populations is consistent with the fact that spatial variation occurs in the temporal cycling of phenotypes; such processes contribute in generating a geographic mosaic in this coevolutionary interaction on the landscape scale. Comparisons of contemporary plant phenotype distributions with phenotypes of herbarium specimens collected 9-125 years ago from across a similar latitudinal gradient, however, suggest that for at least one resistance trait-sphondin concentration-interactions with webworms have led to escalatory change.

Physiological price of an induced chemical defense: photosynthesis, respiration, biosynthesis, and growth.

A recurring theme in defense allocation theories is that defenses are costly. Most studies that attempt to quantify a cost of defense seek to establish a trade-off between a component of plant fitness and the level of a constitutive defense. Such estimates are ambiguous because they cannot discount the cost of traits that are correlated with defense but are not themselves defensive. We examined the effects of damage-induced synthesis of furanocoumarins, known defense compounds, on the growth of wild parsnip. Plants that had 2% of their leaf area removed accumulated 8.6% less total biomass and 14% less root biomass than intact plants over a 4-week period. We also found that this small amount of leaf damage significantly reduced net photosynthetic rates 0.5 h after damage; the effect was temporary, as photosynthetic rates were no longer significantly different after 48 h. Lastly, we found that increases in respiration rates associated with damage coincided spatially and temporally with increases in furanocoumarin production, and that respiration increases were phenotypically correlated with furanocoumarin production. When damage-induced changes in furanocoumarin content and respiration rates were expressed in glucose equivalents and compared, the energetic cost of furanocoumarin production (12.6 µg glucose cm-2) accounted for all of the increase in respiration (12.0 µg glucose cm-2). A comparison of other secondary compounds in damaged and intact leaflets revealed that myristicin, a furanocoumarin synergist, is the only other compound aside from furanocoumarins that is inducible. The inducible defense system of wild parsnip thus appears to involve a small subset of secondary compounds. Synthesis of these compounds is tightly linked to damage-induced rates of respiration. Because the negative impact that damage had on the rate of net photosynthesis was short-lived, the impact of damage on growth observed in this study was likely due to the cost of furanocoumarin synthesis elicited by damage rather than the loss of photosynthetic tissue caused by damage.

Furanocoumarin metabolism in Papilio polyxenes: biochemistry, genetic variability, and ecological significance.

The ubiquitous occurrence of series of biosynthetically related plant secondary compounds within individual species has given rise to the suggestion that such multiplicity is adaptive; one possible mechanism that would serve to maintain such within-plant diversity is analog synergism. In a series of experiments, we provide evidence that synergism may account for the presence of multiple structurally related furanocoumarins in apiaceous plants. The black swallowtail, Papilio polyxenes, feeds exclusively on plant species containing furanocoumarins. Growth of larvae fed parsley leaves treated with both xanthotoxin and angelicin, two furanocoumarins that co-occur widely in swallowtail hostplants, was significantly slower than that of larvae fed leaves with an equimolar concentration of either xanthotoxin or angelicin. A multivariate combination of growth, food consumption and frass excretion differed significantly between larvae fed leaves treated with both xanthotoxin and angelicin and larvae fed leaves treated with angelicin alone. In addition, we measured rates of in vitro cytochrome P450-mediated metabolism of three furanocoumarins - bergapten, xanthotoxin, and angelicin. While bergapten and xanthotoxin, both linear furanocoumarins, were metabolized at similar rates (8.07 and 9.86 nmoles/min/g fw caterpillar, respectively), angelicin, an angular furanocoumarin, was metabolized more slowly (2.76 nmoles/min/g fw caterpillar). When all three furanocoumarins were assayed together, overall rates of metabolism were significantly reduced, suggesting substrate inhibition. Thus, the pattern of growth of larvae is consistent with the pattern of in vitro metabolism and is evidence in support of analog synergism. In a separate experiment, metabolism of xanthotoxin and angelicin individually and together were compared in six maternal families. Again, angelicin was metabolized more slowly than xanthotoxin and each furanocoumarin inhibited metabolism of the other. That significant family effects were found for rates of metabolism and for the ratio of moles of angelicin metabolized for each mole of xanthotoxin metabolized raises the possibility that genetic variation exists for the rate and specificity of metabolism and suggests that insect herbivores may be able to adapt to analog synergism.

GENETICS OF PHYSIOLOGICAL AND BEHAVIORAL RESISTANCE TO HOST FURANOCOUMARINS IN THE PARSNIP WEBWORM.

Depressaria pastinacella, the parsnip webworm, feeds almost exclusively on the flowers and fruits of Pastinaca sativa, the wild parsnip. Resistance to webworms in wild parsnip populations is largely attributable to genetically based variation in furanocoumarin chemistry; by differentially reducing fruit set among chemical phenotypes, parsnip webworms may act as selective agents on wild parsnip populations. To determine whether wild parsnip chemistry can act as a selective agent on webworm populations, it is necessary to establish that resistance mechanisms in the webworm to furanocoumarins are genetically based. In this study, we estimated the amount of genetic variation in behavioral and physiological responses of webworms to parsnip furanocoumarins. Virtually no variation was found among webworm families for feeding preferences for diets varying as much as fourfold in furanocoumarin content. Nor was significant variation found for mean furanocoumarin intake over the assay period, except in one case, in which maternal effects may account for differences among families. In contrast, substantial familial variation existed for cytochrome P450-mediated metabolism of bergapten and xanthotoxin, two host furanocoumarins. The presence of additive genetic variation in metabolism, and the absence of such variation in discriminative feeding behavior, suggests that adaptation to changes in furanocoumarin chemistry, resulting either from changes in the distribution of chemical phenotypes in parsnip populations or from shifts to new chemically different host plants, is likely to be facilitated by physiological rather than behavioral means.