Papilio Polyxenes Research Articles

Convergent evolution of neuroendocrine control of phenotypic plasticity in pupal colour in butterflies

Phenotypic plasticity in pupal colour occurs in three families of butterflies (the Nymphalidae, Papilionidae and Pieridae), typically in species whose pupation sites vary unpredictably in colour. In all species studied to date, larvae ready for pupation respond to environmental cues associated with the colour of their pupation sites and moult into cryptic light (yellow–green) or dark (brown–black) pupae. In nymphalids and pierids, pupal colour is controlled by a neuroendocrine factor, pupal melanization‐reducing factor (PMRF), the release of which inhibits the melanization of the pupal cuticle resulting in light pupae. In contrast, the neuroendocrine factor controlling pupal colour in papilionid butterflies results in the production of brown pupae. PMRF was extracted from the ventral nerve chains of the peacock butterfly Inachis io (Nymphalidae) and black swallowtail butterfly Papilio polyxenes (Papilionidae). When injected into pre‐pupae, the extracts resulted in yellow pupae in I. io but brown pupae in P. polyxenes . These results suggest that the same neuroendocrine factor controls the plasticity in pupal colour, but that plasticity in pupal colour in these species has evolved independently (convergently).

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

Xanthotoxin, a plant allelochemical, induces α-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 α-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 (SRS1) 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 α-cypermethrin.

Tyramine from the leaves of wild parsnip: a stimulant and synergist for oviposition by the black swallowtail butterfly

Tyramine from the leaves of wild parsnip: a stimulant and synergist for oviposition by the black swallowtail butterfly

Tyramine from the leaves of wild parsnip: a stimulant and synergist for oviposition by the black swallowtail butterfly

Abstract.Oviposition stimulants from the foliage of wild parsnip, Pastinaca sativa (Apiaceae), were isolated by column chromatography and HPLC and tested in bioassay experiments with hand‐held female black swallowtail butterflies, Papilio polyxenes (Lepidoptera: Papilionidae). Two of the stimulants were identified as tyramine and trans‐chlorogenic acid. A combination of tyramine, trans‐chlorogenic acid and an active neutral fraction was needed to elicit a significant oviposition response. These results are discussed in the context of previous research on the oviposition stimulants of swallowtail butterflies and on the significance of tyramine as a neuromodulator of physiological processes in invertebrates.

The evolution of environmentally‐cued pupal colour in swallowtail butterflies: natural selection for pupation site and pupal colour

1. Environmentally‐cued pupal colour in swallowtail butterflies has been hypothesized to evolve as a consequence of (a) the evolution of a preference for pupation sites above the ground that vary in colour and (b) natural selection for crypsis on such sites.2. This hypothesis was tested by comparing the field survival of green and brown Papilio polyxenes Fabr. pupae placed on green or brown pupation sites that were either above the ground on near the ground.3. Green pupae on green sites above the ground had a significantly higher probability of survival than did all other pupal colour and pupation site combinations.4. Pupae on sites above the ground were more likely to be preyed upon during the day, whereas those on sites near the ground were more likely to be preyed upon during the night, suggesting that variation in nocturnal and diurnal predation influences the evolution of pupation site preference.5. To the extent that diurnal predators use colour vision to locate prey, diurnal predation should favour environmentally‐cued pupal colour.

Cytochrome P450 (CYP)9A1 in Heliothis virescens: the first member of a new CYP family

A novel cytochrome P450 cDNA with its complete coding sequence and part or all of the 3′ (77 nucleotides) and 5′ (87 nucleotides) non-coding sequence was isolated from the tobacco budworm, Heliothis virescens (F). The 1763 nucleotide sequence encodes a protein of 532 amino acids which includes a hydrophobic N-terminal region and the highly conserved heme binding regions typical of P450s. Low sequence similarity to other P450 sequences and the presence of a thromboxane synthase-like insertion upstream from the I helix resulted in its assignment as the first member of family 9, i.e. CYP9A1. CYP9A1 is most similar to CYP3A1 from the rat (34.7% identity), but is also similar to the insect P450s from family 6, including CYP6BIv1 from Papilio polyxenes (33.3%), CYP6A2A from Drosophila melanogaster (32.4%), CYP6A3 from Musca domestica (31.7%) and CYP6B2 from Helicoverpa armigera (30.1%). Comparative Western and Northern blot studies indicate that expression of CYP9A1 in thiodicarb selected populations of tobacco budworm is associated with insecticide resistance. The pattern of restriction fragment length polymorphism (RFLP) variation in offspring of singlepair matings demonstrated autosomal inheritance of CYP9A1 and enabled its assignment to linkage group 7. The coding region of CYP9A1 occupies no more than 10 kb in the tobacco budworm genome.

Isolation and characterization of CYP6B4, a furanocoumarin-inducible cytochrome P450 from a polyphagous caterpillar (Lepidoptera: Papilionidae)

Papilio glaucus (tiger swallowtail) is a generalist that rarely encounters plants containing furanocoumarins yet is constitutively capable of metabolizing low levels of these highly toxic allelochemicals. In larvae of this species, metabolism of linear (xanthotoxin, bergapten), and angular (angelicin, sphondin), furanocoumarins can be induced up to 30-fold by the presence of xanthotoxin in their diet. Degenerate primers corresponding to conserved amino acid sequences in three insect P450s, Musca domestica (CYP6A1), Drosophila melanogaster ( CYP6A2) and Papilio polyxenes (CYP6B1) , were used to clone xanthotoxin-induced P450 transcripts from P. glaucus larvae by a reverse transcription-polymerase chain reaction (RTPCR) strategy. Positive clones encoding the highly conserved F--G-R-C-G P450 signature motif were used to isolate a full-length CYP6B4v1 cDNA from a P. glaucus xanthotoxin-induced cDNA library. Sequence comparisons indicate the P. glaucus CYP6B4v1 protein sequence is 63% and 61% identical, respectively, to the P. polyxenes furanocoumarin-inducible CYP6Blv1 and CYP6B3v1 proteins. Northern analysis indicates that CYP6B4 and related transcripts are highly induced in response to xanthotoxin. Baculovirus-mediated expression of the CYP6B4v1 protein in lepidopteran cell lines demonstrates that this P450 isozyme metabolizes isopimpinellin, imperatorin, and bergapten at high rates, xanthotoxin and psoralen at intermediate rates and angelicin, sphondin, and trioxsalen only at very low rates.

Quantification of contact oviposition stimulants for black swallowtail butterfly,Papilio polyxenes, on the leaf surfaces of wild carrot,Daucus carota

Ovipositing black swallowtail butterflies,Papilio polyxenes, make their final host-selection decisions on the basis of compounds present on the leaf surface. Little information is available, however, on the chemistry of leaf surfaces. The purpose of this study was to develop a technique to extract and quantify the concentrations of compounds from the leaf surfaces ofDaucus carota, one of the main host species forP. polyxenes, with particular reference to compounds already identified as contact oviposition stimulants, namelytrans-chlorogenic acid (CA) and luteolin-7-O-(6″-O-malonyl)-β-D-glucopyranoside (L7MG), as well as its degradation product luteolin-7-glucoside (L7G). Plant surfaces were extracted by dipping leaves sequentially in pairs of solvents: (1) CHCl3-MeOH, (2) near-boiling H2O, (3) CHCl3-near-boiling H2O, and (4) CH2Cl2-CH2Cl2. The resulting extracts were fractionated and analyzed using high-performance liquid chromatography. The leaf-surface concentrations of each compound were calculated using regressions relating leaf surface area to leaf weight that were obtained from measurements of field-collected carrot plants. All four methods removed the three compounds from carrot leaf surfaces, but the solvent systems differed in effectiveness. The chloroform-near-boiling water solvent system performed better than the other solvent combinations, but not significantly so. This system also extracted the highest number of polar, UV-absorbing compounds. Methylene chloride was significantly less efficient than the other methods. An additional test confirmed that the chloroform-near-boiling water method removed compounds from the surface alone and probably not from the apoplast or symplast. Surface concentrations of CA (up to 600 ng/cm(2) leaf surface) were substantially greater than those of the two flavonoid compounds. No clear seasonal trend in concentrations was evident from the limited number of sampling dates.

Conserved promoter elements in the CYP6B gene family suggest common ancestry for cytochrome P450 monooxygenases mediating furanocoumarin detoxification.

Despite the fact that Papilio glaucus and Papilio polyxenes share no single hostplant species, both species feed to varying extents on hostplants that contain furanocoumarins. P. glaucus contains two nearly identical genes, CYP6B4v2 and CYP6B5v1, and P. polyxenes contains two related genes, CYP6B1v3 and CYP6B3v2. Except for CYP6B3v2, the substrate specificity of which has not yet been defined, each of the encoded cytochrome P450 monooxygenases (P450s) metabolizes an array of linear furanocoumarins. All four genes are transcriptionally induced in larvae by exposure to the furanocoumarin xanthotoxin; several are also induced by other furanocoumarins. Comparisons of the organizational structures of these genes indicate that all have the same intron/exon arrangement. Sequences in the promoter regions of the P. glaucus CYP6B4v2/CYP6B5v1 genes and the P. polyxenes CYP6B3v2 gene are similar but not identical to the -146 to -97 region of CYP6B1v3 gene, which contains a xanthotoxin-responsive element (XRE-xan) important for basal and xanthotoxin-inducible transcription of CYP6B1v3. Complements of the xenobiotic-responsive element (XRE-AhR) in the dioxin-inducible human and rat CYP1A1 genes also exist in all four promoters, suggesting that these genes may be regulated by dioxin. Antioxidant-responsive elements (AREs) in mouse and rat glutathione S-transferase genes and the Barbie box element (Bar) in the bacterial CYP102 gene exist in the CYP6B1v3, CYP6B4v2, and CYP6B5v1 promoters. Similarities in the protein sequences, intron positions, and xanthotoxin- and xenobiotic-responsive promoter elements indicate that these insect CYP6B genes are derived from a common ancestral gene. Evolutionary comparisons between these P450 genes are the first available for a group of insect genes transcriptionally regulated by hostplant allelochemicals and provide insights into the process by which insects evolve specialized feeding habits.

INTRASEXUAL SELECTION CONSTRAINS THE EVOLUTION OF THE DORSAL COLOR PATTERN OF MALE BLACK SWALLOWTAIL BUTTERFLIES, PAPILIO POLYXENES.

Males of the eastern black swallowtail (Papilio polyxenes asterius Stoll) with typical coloration were more successful in intrasexual competition for mating territories than were males altered to have female-like mimetic coloration. Sibling males were matched for wingspan and emergence date and released as pairs, one with its pattern altered and one a control that was marked but with unaltered appearance. Significantly fewer altered males were resighted one or more days after release compared with control males (33% vs. 76%, 1990; 46% vs. 83%, 1993). Altered males were less able to establish and maintain themselves in preferred territories. The inability of released, altered males to establish a territory appears related to significantly longer male-male encounters. Encounters involving at least one participant with altered appearance averaged 66 s compared with 24 s if neither male was altered. However, altering the coloration of P. polyxenes males that already had established themselves in a territory had little effect. After courtships of similar duration (≈ 40 s), released virgin females were equally likely to mate with either altered or control males. This suggests that male-male intrasexual selection is of greater importance than female mate choice in maintaining a non-mimetic dorsal coloration in male P. polyxenes.

Pupation site preference and environmentally cued pupal colour dimorphism in the swallowtail butterfly Papilio polyxenes Fabr. (Lepidoptera: Papilionidae)

Environmentally cued polymorphisms are hypothesized to evolve when the environment is coarse-grained and different genotypes are unable to choose the habitats in which they are most fit. InPapilio polyxenes, which has an environmentally cued pupal colour dimorphism, there is genetic variation in both tendency to produce brown or green pupae and preference for green- or brown-inducing pupation sites, but the two traits are not correlated.

Isolation and sequence analysis of a cDNA clone for a pyrethroid inducible cytochrome P450 from Helicoverpa armigera

The complete coding sequence and parts of the 3′ and 5′ noncoding regions of a mRNA coding for a cytochrome P450 from Helicoverpa armigera have been obtained. The sequence is most similar to members of family CYP6, in particular that obtained from Papilio polyxenes, CYP6B1, and has been labeled CYP6B2 accordingly. The original cDNA was obtained by screening a cDNA library with an oligonucleotide specific for the amino acid sequence surrounding the cysteine residue involved in heme binding, present in the other known insect sequences. This sequence is also present in mammalian members of family CYP3. The highly conserved nature of this particular sequence suggests that this approach may allow the easy and direct identification of cDNA clones specific for members of this particular cytochrome P450 family from a wide variety of species of invertebrates and, possibly, vertebrates. The cDNA hybridizes to two major mRNAs of 2.1 and 1.8 kb in length. Induction studies indicate that the smaller mRNA is inducible by phenobarbital while the larger mRNA is inducible by the pyrethroid insecticide permethrin. Both the evolution of this particular family of cytochrome P450 genes and its potential involvement in the development of resistance to pyrethroids is discussed.

CYP6B3: a second furanocoumarin‐inducible cytochrome P450 expressed in Papilio polyxenes

Cytochrome P450 monooxygenases in the larvae of Papilio polyxenes (black swallowtail) (Lepidoptera: Papilionidae) are capable of detoxifying linear and angular furanocoumarins found in their host plants. The CYP6B1 locus, which is transcriptionally induced in these larvae in response to xanthotoxin, encodes a P450 that principally metabolizes linear furanocoumarins such as xanthotoxin and bergapten. We have now cloned CYP6B3 cDNA derived from a second locus that is evolutionarily related to the CYP6B1 locus. Reverse transcription-PCR Southern analyses have demonstrated that CYP6B3 transcripts are expressed in response to a wider range of linear and angular furanocoumarins but at lower abundance than CYP6B1 transcripts. Whereas CYP6B1 transcripts are expressed at a low detectable level in uninduced control larvae and at high levels in xanthotoxin-induced larvae, CYP6B3 transcripts are nearly undetectable in control larvae and are highly induced by xanthotoxin and bergapten (linear furanocoumarins) as well as by angelicin and sphondin (angular furanocoumarins). The fact that these two CYP6B loci are differentially regulated by these four furanocoumarins indicates that P. polyxenes has adapted to the presence of the wide range of furanocoumarins in its host plants by diversifying its P450 isozyme structure and its furanocoumarin-responsive regulatory cascades.

Female-limited variability in mimicry in the swallowtail butterfly Papilio polyxenes Fabr.

Variation in dorsal and ventral aspects of mimetic wing patterns was measured in the sexually dimorphic swallowtail butterfly Papilio polyxenes, a Batesian mimic of Battus philenor. The pattern on the dorsal surface of the wings makes females better mimics than males and was more variable in females. The pattern on the ventral surface of the hind wings, which provides a similar degree of mimicry in both sexes, was equally variable in both sexes. Full-sib ANOVA indicated that variability in dorsal and ventral patterns was similarily heritable in both sexes, suggesting that the increased variability in females is not simply the result of suppressed expression of genetic variation in males. The results are consistent with the hypothesis that variation in two sets of modifier loci, only one of which is expressed in males, influences the dorsal pattern, resulting in greater variability in females. The nonmimetic dorsal pattern in males is probably maintained as a consequence of sexual selection arising from male-male competition in territorial encounters. The generality of the reduced male variability in the Lepidoptera is discussed.

Differential induction of cytochrome P450 transcripts in Papilio polyxenes by linear and angular furanocoumarins

Cytochrome P450 monooxygenases play critical roles in the detoxification of linear and angular furanocoumarins present in the host plants of Papilio polyxenes (black swallowtail) larvae. To determine the spectrum of linear and angular furanocoumarins that induce isozymes responsible for these detoxifications, the metabolic activities of individual P. polyxenes larvae were monitored in response to individual furanocoumarins. Two linear furanocoumarins, xanthotoxin and bergapten, as well as the angular furanocoumarin, angelicin, significantly and selectively induce metabolism of several furanocoumarins. Another angular furanocoumarin, sphondin, does not induce furanocoumarin metabolism even though it is itself capable of being metabolized in vivo. Northern analysis of the mRNAs induced in response to each of four naturally occurring furanocoumarins indicates that mRNAs related to CYP6B1 are highly induced by the two linear furanocoumarins (xanthotoxin, bergapten) and by angelicin, the angular furanocoumarin. RNase protection analysis indicates that CYP6Blv1–3 transcripts, which we have already cloned, are specifically induced by xanthotoxin and that additional CYP6Bl-related transcripts are induced in response to angelicin. These data indicate that a complex array of CYP6B1 transcripts, derived from a minimum of two loci, is responsible for furanocoumarin metabolism in P. polyxenes. Transcripts derived from these loci appear to be differentially induced by linear and angular furanocoumarins.

Black Swallowtail ( Papilio polyxenes) Alleles Encode Cytochrome P450S That Selectively Metabolize Linear Furanocoumarins

Cytochrome P450 monooxygenases are involved in metabolism of hostplant allelochemicals by larval Lepidoptera. Biochemical purification of the P450 polypeptide induced in Papilio polyxenes (black swallowtail) larvae in response to xanthotoxin, a linear furanocoumarin, has allowed us to clone cDNAs encoding two allelic variants of the CYP6B1 locus. Expression of these alleles in lepidopteran cell lines using baculovirus expression vectors has demonstrated that both P450 isoforms metabolize substantial amounts of linear furanocoumarins, such as xanthotoxin and bergapten, but not angular furanocoumarins, such as angelicin and sphondin. These linear furanocoumarins are ubiquitous constituents of the hostplants of P. polyxenes. The efficiency of linear furanocoumarin metabolism is strongly affected by the nature of the substituents on the benzene ring; methoxy-derivatives are metabolized more efficiently than are derivatives with smaller (hydroxy-) or larger (8- O isopentenyl) groups. Metabolism of either bergapten or xanthotoxin is inhibited in the presence of the other. In addition, metabolism of linear furanocoumarins is inhibited by the presence of nonmetabolizable angular furanocoumarins, indicating that the active site of CYP6B1 binds angular furanocoumarins. The reactivities described here indicate that P. polyxenes larvae express at least two selective furanocoumarin-metabolic P450s: CYP6B1, which metabolizes a discrete set of linear furanocoumarins, and another P450, as yet unidentified, which metabolizes angular furanocoumarins more efficiently than does CYP6B1.

Transcriptional regulation of the Papilio polyxenes CYP6B1 gene.

Detoxification of host plant defensive compounds by larval Lepidoptera is mediated by cytochrome P450 monooxygenases (P450s) such as CYP6B1, which is expressed in Papilio polyxenes (black swallowtail) larvae in response to xanthotoxin, a linear furanocoumarin. Baculovirus-mediated expression of two cloned CYP6B1 cDNAs in lepidopteran cell lines has demonstrated that CYP6B1 isozymes primarily metabolize the linear furanocoumarins, xanthotoxin and bergapten, and not angular furanocoumarins. To characterize the regulatory features of the CYP6B1 transcription unit, we have isolated the first full-length CYP6B1v3 genomic DNA clone from P. polyxenes. The open reading frame of this gene is interrupted by a single intron and is virtually identical to the previously characterized CYP6B1 cDNAs. Primer extension and ribonuclease protection analyses have localized the transcription initiation site to a point 28 nucleotides upstream from the AUG initiation codon. RNase protection analyses on RNA from larvae induced by linear and angular furanocoumarins indicate that transcription of the CYP6B1 gene is induced in insects significantly in response to xanthotoxin and only slightly in response to bergapten. Angular furanocoumarins, such as angelicin, which are not appreciably metabolized by the CYP6B1 gene product, do not significantly induce transcription of this gene. We conclude that this P450 gene is transcriptionally regulated in vivo by at least one of the substrates which the encoded protein metabolizes. Transient expression of CAT fusion constructs in transfected Sf9 lepidopteran cells demonstrates that nucleotides -1 to -838 upstream from the CYP6B1v3 transcription initiation site retain basal and xanthotoxin-inducible transcriptional activities in this heterologous cell line. These data clearly indicate that P. polyxenes has adapted to the presence of furanocoumarins in its host plants by evolving P450 isozymes and regulatory cascades which respond to specific toxins.

Cytochromes P450 from Papilio polyxenes: Adaptations to host plant allelochemicals

Abstract 1. Papilio polyxenes , a caterpillar which feeds on xanthotoxin-containing plants, has cytochromes P450 that are six- to 100-fold less sensitive to the suicide substrate inhibitor, xanthotoxin, than cytochromes P450 from Manduca sexta , which does not survive on xanthotoxin-containing plants. 2. Xanthotoxin is a suicide substrate inhibitor of O -demethylation of p -nitroanisole by M. sexta microsomes but a reversible inhibitor of O -demethylation by P. polyxenes microsomes. 3. Aldrin epoxidation is irreversibly inhibited by xanthotoxin in both species. 4. Patterns of cross inhibition demonstrate that O -demethylase and aldrin epoxidase from both species and the P. polyxenes xanthotoxin-metabolizing cytochrome P450 are distinct enzymes.

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.

Oviposition stimulants for the black swallowtail butterfly: Identification of electrophysiologically active compounds in carrot volatiles

Headspace volatiles were collected from undamaged foliage of carrot,Daucus carota, a host-plant species of the black swallowtail butterfly,Papilio polyxenes. The volatiles were fractionated over silica on an open column, and the fractions were tested in behavioral assays withP. polyxenes females in laboratory experiments. The polar fractions, as well as the total mixture of volatiles, increased the landing frequency and the number of eggs laid on model plants with leaves bearing contact-oviposition stimulants. The nonpolar fraction, containing the most abundant compounds in carrot odor, was not stimulatory. Gas Chromatographic (GC) separation of the fractions was coupled with electroantennogram (EAG) recordings to identify the compounds perceived byP. polyxenes females. The EAG activity corresponded to the behavioral activity of the fractions. None of the nonpolar compounds, identified as various monoterpenes, evoked a major EAG response, but several constituents of the polar fractions elicited high EAG responses. Sabinene hydrate (both stereoisomers), 4-terpineol, bomyl acetate, and (Z)-3-hexenyl acetate were identified by GC-MS as active compounds.