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.     

Ecology and genetics of interspecific hybridization in the swallowtails, Papilio hospiton Gene and P. machaon L., in Corsica (Lepidoptera: Papiliomdae)

The Corsican swallowtail butterfly, Papilio hospiton , is endemic to Corsica and Sardinia (France and Italy) and included in the list of endangered species by the Washington Convention, It is spread all over Corsica in scattered populations linked to diverse habitats. A study by enzyme electrophoresis showed that the genetic diversity of the species is of the same order of magnitude as that of Papilio machaon from continental France and Corsica. The differentiation between populations is rather low, which is consistent with the high vagility of the adults. Natural hybridization between P. hospiton and P. machaon is frequent, and laboratory crosses show that the hybrids are not sterile. However, developmental perturbations impair the viability of further hybrid progenies. Although limited introgression between the two species is likely to take place, enzyme electrophoresis and PCR-RFLP analysis of mitochondrial DNA show that their gene pools remain distincfTtienetic assimilation by P. machaon therefore does not seem to be a threat for P. hospiton.     

Chemical barriers to adaptation by a specialist herbivore

Summary Depressaria pastinacella, the parsnip webworm (Lepidoptera: Oecophoridae), feeds throughout eastern North America on Pastinaca sativa (wild parsnip) and few other species. The assumption that specialist herbivores such as the parsnip webworm are adapted to hostplant chemistry, and are therefore unaffected by chemical variation in hostplants, was tested. Flower buds from plants grown first in the greenhouse and then in the field were fed to ultimate instar webworms. Plant phenotype had a significant effect on virtually all webworm food utilization parameters. While nutritional factors (i.e., nitrogen content) were correlated with approximate digestibility, two constituents of the flowers — bergapten and xanthotoxin, both linear furanocoumarins — independently accounted for a significant amount of variation in food utilization indicies. The physiological effects of these furanocoumarins were confirmed in artificial diet experiments. Despite the fact that the two most important furanocoumarins in parsnip flowers relative to webworm feeding and growth are isomers, differing only in the positioning of a methoxy substituent, they have different physiological effects; while xanthotoxin in general has no effect on growth, bergapten decreases growth and digestibility of the diet. These results underscore the need in studies of plant-animal interactions to examine individual chemical components rather than classes of compounds.     

Morphology of foretarsal ventral surfaces of Japanese Papilio butterflies and relations between these morphology, phylogeny and hostplant preferring hierarchy

Foretarsal ventral surface observation of Japanese Papilio butterflies showed that the shapes of fifth foretarsi and numbers and localization of contact chemosensilla and spines in these areas are closely related to both phylogeny and behavior in these species. My results basically supported the classification that Japanese Papilio divides into five subgenera -- Papilio (P. machaon), Princeps (P. xuthus and P. demoleus), Achillides (P. maackii and P. bianor), Menelaides (P. helenus, P. polytes, P. protenor and P. macilentus) and Iliades (P. memnon). Moreover, female foretarsal morphology also corresponded to the physical features of their preferring host plant leaves. The specificity of female P. machaon, female P. macilentus and female P. maackii seemed to also relate to the geographical diversities of these species and their hostplant, and their co-evolution.     

Molecular analysis of CYP321A1, a novel cytochrome P450 involved in metabolism of plant allelochemicals (furanocoumarins) and insecticides (cypermethrin) in Helicoverpa zea

Cytochrome P450 monooxygenases play a significant role in the detoxification of hostplant allelochemicals and synthetic insecticides in Lepidoptera. In the corn earworm Helicoverpa zea, a noctuid of considerable economic importance, metabolisms of xanthotoxin, a toxic furanocoumarin, and alpha-cypermethrin, an insecticide, are mediated by at least one P450 with a catalytic site capable of accepting both substrates. To further the characterization of P450s in this species, we have cloned three full-length cDNAs encoding two CYP4M subfamily members and a novel CYP321A subfamily member. RNA analyses have demonstrated that the CYP321A1 gene is highly induced (51-fold) in larval midguts in response to xanthotoxin but not cypermethrin. Both CYP4M genes are expressed at negligible levels that are not increased by xanthotoxin or cypermethrin. Baculovirus-mediated expression of the full-length CYP321A1 cDNA has demonstrated that the CYP321A1 protein metabolizes xanthotoxin and angelicin, like the CYP6B1 protein in the furanocoumarin specialist Papilio polyxenes, and alpha-cypermethrin, like the CYP6B8 protein previously characterized in H. zea. In contrast, the CYP4M7 protein does not metabolize xanthotoxin at any detectable level. We conclude that at least two xanthotoxin-inducible P450s from highly divergent subfamilies (CYP6B and CYP321A) contribute to the resistance of H. zea larvae to toxic furanocoumarins and insecticides. Genomic PCR analysis indicates that the CYP321A1 gene has evolved independently from the CYP6B genes known to be present in this insect.     

Differential patterns of hybridization and introgression between the swallowtails Papilio machaon and P. hospiton from Sardinia and Corsica islands (Lepidoptera,Papilionidae)

Proportions of hybridization and introgression between the swallowtails Papilio hospiton, endemic to Sardinia and Corsica, and the holarctic Papilio machaon, were characterized using nine fully diagnostic and two differentiated allozyme loci and a mitochondrial DNA marker. Very low frequencies of F1 hybrids were detected in both Sardinia (0-4%, average 1.4%) and Corsica (0-3%, average 0.5%), as well as of first generation backcrosses (B1). No F2 were observed, in agreement with the hybrid breakdown detected in laboratory crosses. In spite of this minimal current gene exchange, specimens carrying introgressed alleles were found in high proportions in P. machaon but in lower proportions in P. hospiton. Introgression apparently occurred through past hybridization and repeated backcrossing, as evidenced by hybrid index scores and Bayesian assignment tests. Levels of introgression were low (0-1%) at two sex-linked loci and mitochondrial DNA, limited (0.4-2%) at three autosomal loci coding for dimeric enzymes, and high (up to 43%) at four autosomal loci coding for monomeric enzymes. Accordingly, selective filters are acting against foreign alleles, with differential effectiveness depending on the loci involved. The low levels of introgression at sex-linked loci and mitochondrial DNA are in agreement with Haldane's rule and suggest that introgression in P. machaon proceeds mainly through males, owing to a lower fitness of hybrid females. Papilio machaon populations showed higher levels of introgression in Sardinia than in Corsica. The role of reinforcement in the present reproductive isolation between P. machaon and P. hospiton is examined, as well as the evolutionary effects of introgressive hybridization between the two species.     

Enemy-free space and habitat-specific host specialization in a butterfly

The majority of herbivorous insects have relatively specialized food habits. This suggests that specialization has some advantage(s) over generalization. Traditionally, feeding specialization has been thought to be linked to digestive or other food-related physiological advantages, but recent theory suggests that generalist natural enemies of herbivorous insects can also provide a major selective pressure for restricted host plant range. The European swallowtail butterfly Papilio machaon utilizes various plants in the Apiaceae family as hosts, but is an ecological specialist being monophagous on Angelica archangelica in southern Sweden. This perennial monocarp grows in three seaside habitat types: (1) on the barren rocky shore in the absence of any surrounding vegetation, (2) on the rocky shore with some surrounding vegetation, and (3) on species-rich meadows. The rocky shore habitat harbors few invertebrate generalist predators, whereas a number of invertebrate predators abound in the meadowland habitat. Here, we test the importance of enemy-free space for feeding specialization in Papilio machaon by assessing survival of larvae placed by hand on A. archangelica in each of the three habitat types, and by assessing the habitat-specificity of adult female egg-laying behavior by recording the distribution of eggs laid by free-flying adult females among the three habitat types. Larval survival was substantially higher in the rocky shore habitat than in the meadowland and significantly higher on host plants without surrounding vegetation on the rocky shore. Eggs laid by free-flying females were found in all three habitat types, but were significantly more frequent in the rocky shore habitat, suggesting that females prefer to lay eggs in the habitat type where offspring survival is highest. These results show that larval survivorship on the same host plant species can be strongly habitat-specific, and suggest that enemy-free space is an underlying factor that drives feeding specialization in Papilio machaon.     

Identification of Ruta graveolens L. metabolites accumulated in the presence of abiotic elicitors

The study aimed to elucidate the effects of benzothiadiazole (BTH) and saccharin on the biosynthesis of simple coumarins, linear furanocoumarins, dihydrofuranocoumarins, and furoquinolone alkaloids in shoots of R. graveolens cultivated in vitro. The biosynthesized metabolites were analyzed and identified by GC-MS and by comparison of Kovats indices. Eight coumarin metabolites were identified: bergapten, chalepin, isopimpinelin, pinnarin, psoralen, rutacultin, rutamarin, and xanthotoxin, and also four alkaloids: dictamnine, gamma-fagarine, skimmianine, and kokusaginine. Each of the tested BTH concentrations induced a significant production of furanocoumarins and furoquinolone alkaloids. The use of saccharin also increased the production of bergapten, isopimpinelin, pinnarin, psoralen, and xanthotoxin several times.     

Molecular docking of substrates and inhibitors in the catalytic site of CYP6B1, an insect cytochrome p450 monooxygenase

Furanocoumarins represent plant toxins that are used in the treatment of a variety of skin diseases and are metabolized by cytochrome p450 monooxygenases (p450s) existing in insects such as Papilio polyxenes (the black swallowtail). To elucidate the active site in the CYP6B1 protein that is the principal p450 existing in this species, we have constructed a homology model of it based on sequence and structure alignments with the bacterial CYP102 protein whose crystal structure has been defined and with the insect CYP6B4 protein that also metabolizes furanocoumarins. In the derived CYP6B1 model, Phe116 and His117 in SRS1, Phe371 in SRS5 and Phe484 in SRS6 contribute to the formation of a resonant network that stabilizes the p450's catalytic site and allows for interactions with its furanocoumarin substrates. The first two of these residues are absolutely conserved in all members of the insect CYP6B subfamily and the last two are variable in different members of the CYP6B subfamily. A combination of theoretical and experimental docking analyses of two substrates (xanthotoxin and bergapten) and two inhibitors (coumarin and pilocarpine) of this p450 provide significant information on the positioning of furanocoumarins within this catalytic pocket. Molecular replacement models based on the results of variations at two of these critical amino acids provide support for our furanocoumarin-docked model and begin to rationalize the altered substrate reactivities observed in experimental analyses.     

Host plant resistance and linear furanocoumarin content of Apium accessions

Linear furanocoumarin contents and antibiotic resistance to Liriomyza trifolii (Burgess) were documented for Apium species being investigated in a celery breeding program. In no-choice tests, L. trifolii fed more, produced more offspring, and had the highest pupal and adult productivity on the widely planted cultivar 'Tall Utah' 52-70R (Apium graveolens L.). Antibiotic effects of the commercial cultivar 'Tall Utah' 52-70 HK and University of California families 87A-147 and 87A-338, derived from A. chilense Hook and Arn., were intermediate. Only A. nodiflorum (L.) Lag (accession 87A-236) did not allow survival beyond the larval stage. Concentrations of the carcinogenic and mutagenic linear furanocoumarins varied by location within plants (leaves usually greater than petioles), by specific compound (trend: psoralen less than xanthotoxin less than bergapten or isopimpinellin), and between accessions. A. nodiflorum had the lowest foliar levels of phototoxic furanocoumarins (11.8 micrograms/g fresh weight) and the best potential for use in the breeding program. Foliar levels of phototoxic furanocoumarins (psoralen, bergapten, and xanthotoxin) in plants 87A-147-3 (406 micrograms/g), 87A-147-2 (292.9 micrograms/g), and the family 87A-338 (265.9 micrograms/g) were 22.6, 16.3, and 14.8 times higher, respectively, than the concentration known to produce contact dermatitis (18 micrograms/g). Even with such variability in concentration, the foliar content of linear furanocoumarins (individually or total) and L. trifolii adult production were not correlated.     

Papilio phylogeny based on mitochondrial cytochrome oxidase I and II genes

Butterflies of the genus Papilio have served as the basis for numerous studies in insect physiology, genetics, and ecology. However, phylogenetic work on relationships among major lineages in the genus has been limited and inconclusive. We have sequenced 2.3 kb of DNA from the mitochondrial cytochrome oxidase I and II genes (COI and COII) for 23 Papilio taxa and two outgroups, Pachliopta neptunus and Eurytides marcellus, in order to assess the potential of these genes for use in Papilio phylogenetics and to examine patterns of gene evolution across a broad taxonomic range. Nucleotide and amino acid variation is distributed heterogeneously, both within and between genes. Structural features of the proteins are not always reliable predictors of variation. In a combined analysis, these sequences support a nearly fully resolved topology within subgenera and species groups, though higher level relationships among species groups require additional study. The most noteworthy findings are that neither Papilio alexanor nor P. xuthus belongs in the machaon group and that the subgenus Pterourus is paraphyletic with respect to the subgenus Pyrrhosticta. We leave relationships among members of the phorcas species group as a trichotomy. These two protein coding genes, particularly COI, show excellent performance in resolving relationships at the level of species and species groups among Papilionidae. We strongly endorse a similar approach for future studies aimed at these levels.     

Metabolism of linear and angular furanocoumarins by Papilio polyxenes CYP6B1 co-expressed with NADPH cytochrome P450 reductase

One challenge in the heterologous expression of microsomal cytochrome P450 monooxygenases (P450s) is fulfilling their obligatory requirement for electrons transferred from NADPH P450 reductase. We have established co-expression parameters for Papilio polyxenes CYP6B1 and house fly P450 reductase in baculovirus-infected Sf9 cells that allow for efficient expression of both components and significantly enhance metabolic turnover of this insect P450's substrates. These expression conditions have allowed us to reexamine the turnover capacities of CYP6B1 toward linear and angular furanocoumarins present in the host plants for the specialist caterpillar P. polyxenes. Coexpression of CYP6B1 and P450 reductase at equivalent viral concentrations [MOI (multiplicity of infection) ratio of 1] results in turnover rates for the linear furanocoumarins xanthotoxin and psoralen, which are increased 32-33 fold over the turnover rates obtained with CYP6B1 expressed alone. The turnover rate for the angular furanocoumarin angelicin is also significantly increased to 4.76 nmol/min/nmol P450 compared to its barely detectable level obtained with CYP6B1 expressed alone. Substrate binding analyses indicate that all three of these compounds elicit typical type I binding spectra but with varying magnitudes and affinities that are indicative of each substrate's effectiveness at coordinating with the heme iron. The relative proportions of high spin state generated with these substrates are consistent with CYP6B1 metabolizing these furanocoumarins in the rank order xanthotoxin>psoralen>angelicin. These differential activities for CYP6B1 suggest that it may have been an ancient participant in the coevolutionary arms race between papilionid butterflies and their apiaceous host plants. Due to its ability to handle a range of furanocoumarin structures, CYP6B1 may have contributed to P. polyxenes' early colonization of linear furanocoumarin-containing plants and to its subsequent colonization of angular furanocoumarin-containing plants.     

Speciation problems in the Papilio machaon group of butterflies (Lepidoptera: Papilionidae)

Abstract. The taxonomic and genetic relationships between P.machaon rathjensi Warnecke from the west side of southern Arabia and P.machaon muetingi Seyer from the east are discussed, together with suggestions of their relationship to P.machaon saharae Oberthiir from the northern Hejaz, Sinai and desert North Africa. It is concluded that though P.m.rathjensi and P.m.saharae are subspecifically distinct, they are very closely related to each other, and both should be regarded as specifically distinct from the rest of the machaon complex.
The remainder of the machaon complex in its turn presents a somewhat similar problem. Thus P.machaon gorganus Fruhstorfer and P.hospiton Géné are normally regarded as good species, but Fl caterpillars have been found in the wild and the cross can be readily produced in captivity with no upset in the sex ratio. Furthermore, recently we have obtained sparse F2 offspring between the two species.
We therefore suggest that the entire P.machaon complex is in a labile state as regards speciation throughout its range and the relationships between the various forms are not easily expressed through the traditional species and subspecies concepts. Our tentative conclusions are that the status of P.machaon and P.hospiton as distinct species probably remains valid. The saharaelrathjensi complex might be considered distinct from P.machaon using the biological species concept, while their genetics indicate a more conservative approach. However, more important than the exact status of the taxa in question is the fact that the P.machaon complex illustrates evoluton in action, the end result of which cannot be predicted.     

Molecular cloning and expression of CYP6B8: a xanthotoxin-inducible cytochrome P450 cDNA from Helicoverpa zea

Xanthotoxin, a plant allelochemical, induces alpha-cypermethrin insecticide tolerance in Helicoverpa zea (corn earworm); inhibition of tolerance by piperonyl butoxide implicates cytochrome P450 monooxygenases (P450s) in the detoxification of this insecticide. To characterize the xanthotoxin-inducible P450 that might mediate alpha-cypermethrin tolerance in this species, a cDNA library prepared from xanthotoxin-induced H. zea fifth instar larvae was screened with cDNAs encoding furanocoumarin-metabolizing P450s from Papilio polyxenes (CYP6B1v2) and P. glaucus (CYP6B4v2) as well as a sequence-related P450 from Helicoverpa armigera (CYP6B2). One full-length cDNA isolated in this screening shares 51-99% amino acid identity with the CYP6B subfamily of P450s isolated from Papilio and Helicoverpa species and, thus, has been designated CYP6B8. All of these CYP6B subfamily members share a number of highly conserved domains, including substrate recognition site 1 (SRS 1) that is critical for xanthotoxin metabolism by CYP6B1v2 from Papilio polyxenes and coumarin metabolism by CYP2a5 from Mus musculus. Northern and RT-PCR analyses indicate that CYP6B8 expression is strongly induced by xanthotoxin and phenobarbital and negligibly induced by alpha-cypermethrin.     

The phylogeny of some Papilio species based on interspecific hybridization data

Abstract. Adults of different species of the genus Papilio can easily be mated by hand-pairing. The data on egg hatchability, adult formation and F₁ fertility in these interspecific hybrids show notable variations according to the crosses. Using these data, values of a differentiation index for each cross were calculated. These estimates of genetic distance were then used to study the phylo-genetic relationships of the species involved. The data are consistent with the existence of the following groups, each consisting of closely related species: the black Papilio supergroup, the Papilio memnon group, the P.demoleus group, the P.xuthus group, the P.machaon group, the P.bianor group, the P.glaucus group and the P.troilus group. The phylogenetic relationships revealed by these data generally coincide with the relationships established by classical taxonomy. In cases where there are differences of opinion among taxonomists, the writer's data usually support one or other of them.     

Differential response of cultured parsley cells to elicitors from two non-pathogenic strains of fungi. 1. Identification of induced products as coumarin derivatives

Dark-grown cell suspension cultures of parsley, Petroselinum hortense, produce furanocoumarins after treatment with elicitor preparations of either Phytophthora megasperma f.sp. glycinea (Pmg elicitor) or Alternaria carthami Chowdhury (Ac elicitor). The linear furanocoumarins, psoralen and xanthotoxin, and the benzodipyrandione, graveolone, are the major products synthesized in response to Pmg elicitor, besides small amounts of the furanocoumarin bergapten. Treatment with Ac elicitor induces predominantly the formation of bergapten and the furanocoumarin isopimpinellin, as well as small amounts of graveolone. While Pmg elicitor leads to cell death within a few days, cell mass increased for at least 6 days after treatment with Ac elicitor. Brefeldin A, a phytotoxin produced by A. carthami, inhibits growth of parsley cell suspension cultures considerably at a concentration of 0.01 mM and growth of the cells ceased at a concentration of 0.1 mM toxin. Concomitantly, furanocoumarin biosynthesis was suppressed in our system by a concentration of brefeldin A within 0.01-0.1 mM.