Papilio Polyxenes Research Articles

Regulation of an insect cytochrome P450 monooxygenase gene ( CYP6B1) by aryl hydrocarbon and xanthotoxin response cascades

Many organisms respond to toxic compounds in their environment by inducing regulatory networks controlling the expression and activity of cytochrome P450 monooxygenase ( P450s) detoxificative enzymes. In particular, black swallowtail ( Papilio polyxenes) caterpillars respond to xanthotoxin, a toxic phytochemical in their hostplants, by activating transcription of the CYP6B1 promoter via several regions located within 150 nt of the transcription initiation site. One such element is the xenobiotic response element to xanthotoxin (XRE–Xan) that lies upstream of consensus XRE–AhR ( xenobiotic response element to the aryl hydrocarbon receptor) and OCT-1 ( octamer- 1 binding site) element known to be utilized in mammalian aryl hydrocarbon response cascades. Two-plasmid transfections conducted in Sf9 cells have indicated that XRE–Xan, XRE–AhR and a number of other proximal elements, but not OCT-1, are critical for basal as well as xanthotoxin- and benzo[α]pyrene-induced transcription of the CYP6B1 promoter. Four-plasmid transfections with vectors co-expressing the spineless (Ss) and tango (Tgo) proteins, the Drosophila melanogaster homologues of mammalian AhR and ARNT, have indicated that these proteins enhance basal expression of the CYP6B1 promoter but not the magnitude of its xanthotoxin and benzo[α]pyrene induction. Based on these results, we propose that these Drosophila transcription factors modulate basal expression of this promoter in a ligand-independent manner and attenuate its subsequent responses to planar aryl hydrocarbons (benzo[α]pyrene) and allelochemicals (xanthotoxin).

Ile115Leu mutation in the SRS1 region of an insect cytochrome P450 (CYP6B1) compromises substrate turnover via changes in a predicted product release channel

CYP6B1 represents the principal cytochrome P450 monooxygenase responsible for metabolizing furanocoumarins in Papilio polyxenes, an insect that specializes on host plants containing these toxins. Investigations of the amino acids responsible for the efficient metabolism of these plant toxins has identified Ile115 as one that modulates the rate of furanocoumarin metabolism even though it is predicted to be positioned at the edge of the heme plane and outside substrate contact regions. In contrast to previous expression studies conducted under conditions of limiting P450 reductase showing that the Ile115-to-Leu replacement enhances turnover of xanthotoxin and other furanocoumarins, studies conducted at high P450 reductase indicate that the Ile115-to-Leu replacement reduces turnover of these substrates. Further analysis of substrate binding affinities, heme spin state and NADPH consumption rates indicate that, whereas the I115L replacement mutant displays higher substrate affinity and heme spin state than the wild-type CYP6B1 protein, it utilizes NADPH more slowly than the wild-type CYP6B1 protein at high P450 reductase levels. Molecular models developed for the wild-type CYP6B1 and mutant protein suggest that more constricted channels extending from the catalytic site in the I115L mutant to the P450 surface limit the rate of product release from this mutant catalytic site under conditions not limited by the rate of electron transfer from NADPH.

Mosquitoes and the TV—eh?

Among my many character quirks, I seem to be constitutionally unable to travel anywhere out of the ordinary unless there’s a speaking engagement involved. This not only makes for a great deal of family tension over vacation destinations (which almost invariably are university towns, most of which lack the white sand beaches and other amenities my daughter is understandably partial to when thinking of vacations), but it also is the principal reason that for more than 30 years, I never managed to make it to any kind of reunion—high school, college, graduate school, or even summer camp. It never was for want of interest—I always wondered what it might be like to attend a reunion but never could justify the travel involved to satisfy that curiosity. So, when the 30th Reunion Committee of the Yale Class of 1975 invited me to serve as a member of a discussion panel, I jumped at the chance. Never mind that the invitation was to serve on a panel discussion on bioterrorism, a subject area in which I have no distinguished credentials. The extent of my claim to any expertise at all was the fact that I was one of 16 reviewers of a National Research Council report on agricultural bioterrorism (NAS 2002). Although this study had been commissioned by the Board on Agriculture and Natural Resources, on which I served a term and currently serve as chair, the study was actually conducted during the interval when I was neither on the Board nor Chair of the Board—making my connection to agricultural bioterrorism even more tenuous. Although I do work on insects, and although insects could conceivably be involved in acts of agricultural bioterrorism, the insects I work with have little terrorism potential; the black swallowtail, Papilio polyxenes, may pose a threat to America’s parsley crop, but somehow I think our great nation can survive the challenge of steaks with no garnish. It seemed odd to me that a reunion committee thought a discussion of bioterrorism would be appropriate entertainment for returning alumni—but, since I had so little previous reunion experience, I thought maybe my notion that reunions were for getting together with old friends and trying to remember the lyrics to Cat Stevens songs was perhaps out of step with reality. And it turns out that the panel discussion was well attended, at least in part because the other panel discussion that had been organized, on the difficulties faced by Yale alumni living abroad, had to be cancelled because not enough of the panelists, all Yale alumni living abroad, had managed to make it to the reunion. What surprised me most about the reunion, though, was how few of the people I knew really well while I was an undergraduate at Yale attended the reunion, and how many people that I swear I’d never seen before were actually my classmates. Among the classmates I would have liked to see again was Brandon Centerwall, who was briefly the freshman-year roommate of my best friend Robert; Robert’s erratic hours and professed fondness for licit and illicit psychotropic substances eventually drove Brandon to find alternate living arrangements before the end of first semester. After graduating from Yale, Brandon went on to medical school and became an epidemiologist. He gained no small measure of fame in the field thanks to a series of papers linking television-watching with violent behavior (Centerwall 1989, 1992). In his 1989 study, Brandon compared the relationship between television ownership and homicide rates in the United States and Canada from roughly 1945 to 1975 with the homicide rate in South Africa over the same time period, during which time television broadcasts were banned. Whereas homicides declined by 7% in South Africa over the period, they essentially doubled in the United YIKES! Forget jogging! I think watching TV would be healthier for me!

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.

Effects of contact chemistry and host plant experience in the oviposition behaviour of the eastern black swallowtail butterfly

Papilio polyxenes, the eastern black swallowtail butterfly, is a specialist on members of the carrot family, Apiaceae. Females choose food plants for their larvae, laying eggs singly on acceptable host plants, which vary considerably visually and chemically. To allow for fidelity of these vagile females to a single family of host plants in the absence of information transfer across life stages, the response to at least one type of host plant cue, be it visual or chemical, must be innate. Although much work has been done to identify the plant chemicals used by ovipositing swallowtails to recognize their host plants after landing, it has been assumed rather than demonstrated that the response to this cue is innate. We conducted two experiments to test the nature of the black swallowtail's response to contact chemical host plant cues, and a third to examine the role of the larval host plant in adult behaviour. The results of the first experiment verified that females without host plant experience respond specifically to cues from the host plant, Daucus carota, with typical abdomen curling, and not to the nonhost fava bean, Vicia faba. The nondeterrency of bean was also verified. The second experiment demonstrated that this response was unaltered by specific host plant experience: females responded to extracts of hosts, carrot and poison hemlock, Conium maculatum, differentially, but without regard to the plant previously experienced. We also examined effects of deprivation and age in the first experiment, with each having a significant effect; these were controlled for in the second experiment. Finally, feeding on a host plant as a larva did not increase the preference for that plant as an adult. This is the first demonstration that the response to these contact chemical cues is not only innate but unaltered by specific host plant experience.

Conserved regulatory elements in the promoters of two allelochemical-inducible cytochrome P450 genes differentially regulate transcription

CYP6B4, a cytochrome P450 gene from the tiger swallowtail Papilio glaucus, is transcriptionally induced in the midgut by dietary furanocoumarins, plant allelochemicals that can crosslink DNA in their UV-activated form. The CYP6B4 promoter contains an overlapping EcRE/ARE/XRE-xan element similar to that used for basal and xanthotoxin-inducible expression of the CYP6B1 promoter from the black swallowtail Papilio polyxenes. Transfection of the CYP6B4 promoter:CAT reporter construct into Sf9 cells demonstrates that the basal and xanthotoxin-inducible expression levels observed reflect the relative expression levels of this gene in the midguts of tiger swallowtail larvae. Transfections of mutant CYP6B4 promoter constructs into Sf9 cells indicate that the EcRE/ARE/XRE-xan element is necessary for CYP6B4 induction by xanthotoxin but not for its minimal basal expression. In addition to these elements, the CYP6B4 and CYP6B1 promoters also contain putative XRE-AhR elements identical to the aryl hydrocarbon response elements present in mammalian phase I detoxification genes. Transfections of CYP6B4 and CYP6B1 promoters containing EcRE/ARE/XRE-xan and XRE-AhR elements indicate that both are induced significantly by benzo(α)pyrene, an aryl hydrocarbon widespread in the environment, as well as by xanthotoxin, an allelochemical encountered in their hostplants.

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 α-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 α-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.

Seasonal variation in Daucus carota leaf-surface and leaf-tissue chemical profiles

The purpose of this work was to document seasonal changes in leaf-surface and whole-leaf chemistry of Daucus carota cohorts that differed in life-cycle phenology (winter annual, annual, or biennial), with particular focus on compounds that serve as contact oviposition stimulants for Papilio polyxenes, the black swallowtail butterfly. Cohorts of carrot plants exhibiting different life-cycle phenologies were established, and plants from each cohort were sampled periodically over two growing seasons. Extracts of leaf surfaces and leaf tissues were analyzed using HPLC and were compared quantitatively for the content of chlorogenic acid (CA), luteolin-7-O-(6″-O-malonyl)-β-D-glucopyranoside (L7MG), and luteolin-7-glucoside (L7G). The pattern of seasonal variation in L7G and L7MG concentrations in leaf tissues of each cohort followed the predictions of the tissue value hypothesis: flavonoid concentrations are highest in the tissues of new alternate leaves of the reproductive shoot or in new spring foliage. Chlorogenic acid concentrations in leaf tissues increased throughout the growing season. Patterns of leaf-surface seasonal variation did not correlate with patterns in leaf-tissue chemistry for any carrot cohort. Rather, leaf-surface amounts of CA, L7G, and L7MG were highest at the point in the lifecycle when plants were differentiating or bolting.

Identification of variable amino acids in the SRS1 region of CYP6B1 modulating furanocoumarin metabolism

The homology model of Papilio polyxenes CYP6B1 places Ile115, one of two variable amino acids, in the SRS1 of various CYP6B subfamily proteins in close proximity to the heme and Ala113, another variable amino acid, in a more distal position. We have constructed mutant CYP6B1 proteins altered at either of these positions and homology models of each based on multiple alignments with crystallized P450 proteins. The homology models suggest the existence of significant structural diversity in the hydrogen bond network surrounding the heme as a result of single point mutations in SRS1. Mutagenesis of Ile115 or Ala113 to other residues present in the insect CYP6B subfamily indicates that these amino acids control the spin state of the heme and, as a result, the catalytic activity of this monooxygenase. In particular, the I115L mutation significantly increases the spin state of the heme coordinately with 2- to 4-fold increases in its turnover of linear furanocoumarins. Other A113V, A113L, A113Q, and A113E mutations display more variation in their effects but, in each case, strong correlations exist between furanocoumarin turnover and heme spin state. These data demonstrate that variable amino acids in SRS1 of the insect CYP6B subfamily exert dramatic effects on the range of furanocoumarins metabolized, even when they occur in positions potentially distal from the substrate. These effects are possibly mediated through rearrangement of the local hydrogen bond network.

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.

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.

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.

Amino acids in SRS1 and SRS6 are critical for furanocoumarin metabolism by CYP6B1v1, a cytochrome P450 monooxygenase.

CYP6B1v1 is the principal cytochrome P450 monooxygenase (P450) that detoxifies dietary furanocoumarins in the guts of Papilio polyxenes, the black swallowtail caterpillar. Sequence alignments and structure comparisons of CYP6B1v1 with the mouse CYP2A5 and bacterial CYP102 proteins, which are also capable of metabolizing the linear furanocoumarin xanthotoxin (8-methoxypsoralen), suggested that Phe116, His117, Val368 and Phe484 might be active site residues. In a homology model developed for CYP6B1v1, the side chains of Phe116 and His117 located in the B'-C loop of SRS1 are predicted to be positioned above the haem plane, while the side chain of Phe484 located in SRS6 is predicted near the entrance of the catalytic pocket. Site-directed mutagenesis of residues Phe116, His117 and Phe484 indicated that these residues represent several of those that determine this protein's stability and substrate specificity. Whereas all aromatic mutants of Phe116 and Phe484 generated CO-difference spectra with maxima at 450 nm indicative of correctly configured monooxygenases, aromatic mutants of Phe116 exhibited reduced reactivities toward some furanocoumarins and aromatic mutants of Phe484 eliminated all reactivities toward furanocoumarins. All single and double aliphatic mutants of Phe116, His117 and Phe484 and aromatic mutants of His117 generated carbon monoxide (CO) difference spectra with maxima at 420 nm (P420) indicative of incorrectly configured monooxygenases. These studies define residues Phe116, His117 and Phe484 as determinants of this insect P450's catalytic site integrity and residues Phe116 and Phe484 as determinants of its substrate specificity. Conservation of Phe116 and His117 in an array of lepidopteran CYP6B proteins implies that these amino acids serve a similar function in other monooxygenases of the insect CYP6B subfamily.

THE ENVIRONMENTAL AND GENETIC CONTROL OF SEASONAL POLYPHENISM IN LARVAL COLOR AND ITS ADAPTIVE SIGNIFICANCE IN A SWALLOWTAIL BUTTERFLY

Seasonal polyphenism, in which different forms of a species are produced at different times of the year, is a common form of phenotypic plasticity among insects. Here I show that the production of dark fifth-instar caterpillars of the eastern black swallowtail butterfly, Papilio polyxenes, is a seasonal polyphenism, with larvae reared on autumnal conditions being significantly darker than larvae reared on midsummer conditions. Both rearing photoperiod and temperature were found to have individual and synergistic effects on larval darkness. Genetic analysis of variation among full-sibling families reared on combinations of two different temperatures and photoperiods is consistent with the hypothesis that variation in darkness is heritable. In addition, the genetic correlation in larval darkness across midsummer and autumnal environments is not different from zero, suggesting that differential gene expression is responsible for the increase in larval darkness in the autumn. The relatively dark autumnal form was found to have a higher body temperature in sunlight than did the lighter midsummer form, and small differences in temperature were found to increase larval growth rate. These results suggest that this genetically based seasonal polyphenism in larval color has evolved in part to increase larval growth rates in the autumn.Corresponding Editor: T. Kawecki

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.

GM crops go walkies

Two recent reports revive concerns about the implications of large-scale use of genetically modified (GM) crops. The Mexican government announced that GM DNA could be detected in seeds collected from endogenous corn varieties at 15 different locations, the New York Times reports. So far, it's unclear what the source of the DNA is, but experts think it likely that transgenic ‘Bt’ corn has interbred with local plants. Bt corn carries a bacterial toxin gene that protects the plant from the European corn borer pest. Considering that Bt corn, or indeed any GM crop, cannot be grown in Mexico, the findings suggest that geography and plant variety impose smaller barriers for transmission of transgenes than previously anticipated. Bt corn was also a focus of attention in six papers published on-line in PNAS on 14 September (Zangerl et al., Sears et al., Hellmich et al., Pleasants et al., Stanley-Horn et al., Oberhauser et al.). Previous work had indicated that, under laboratory conditions, Bt toxin not only killed European corn borers but also harmless black swallowtail caterpillars. Here, the authors report that Bt corn pollen can contaminate neighboring plants with doses high enough to kill the swallowtail caterpillars. However, the studies also show that Bt toxin rarely exceeds lethal doses in the fields and poses only a minor threat to the ecosystem. Moreover, the harmful Bt corn variant Bt176 is only a prototype – new Bt corn variants are not harmful, and Syngenta, the producer of Bt corn, has already taken Bt176 off the market. J.d.B.

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.

Bt Corn Variety OK for Black Swallowtails

Science NewsVolume 157, Issue 24 p. 372-373 This Week Bt corn variety OK for black swallowtails Susan Milius, Susan MiliusSearch for more papers by this author Susan Milius, Susan MiliusSearch for more papers by this author First published: 01 July 2009 https://doi.org/10.2307/4012349AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume157, Issue24June 2000Pages 372-373 RelatedInformation

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.