Comparative metabolism of [3H]psoralen and [3H]isopsoralen by black swallowtail (Papilio polyxenes Fabr.) caterpillars

The comparative fate of tritiated preparations of a linear furanocoumarin (psoralen) and an angular furanocoumarin (isopsoralen) was determined in last-instar caterpillars of the black swallowtail butterfly (Papilio polyxenes Fabr.). Oral administration of either furanocoumarin at 5 g/g is followed by rapid metabolism, primarily through oxidative cleavage of the furan ring, and the metabolites are rapidly excreted. Isopsoralen is, however, metabolized at a somewhat slower rate than is psoralen, and levels of unmetabolized isopsoralen in body tissues of the treated caterpillars are about three-fold higher. These data are compatible with the hypothesis that a reduced detoxification rate accounts at least in part for the susceptibility ofP. polyxenes caterpillars to the deleterious effect of isopsoralens.     

In vitro metabolism of a linear furanocoumarin (8-methoxypsoralen,xanthotoxin) by mixed-function oxidases of larvae of black swallowtail butterfly and fall armyworm

Studies were made of the comparative in vitro metabolism of [¹⁴C]xanthotoxin and [¹⁴C]aldrin by homogenate preparations of midguts and bodies (carcass minus digestive tract and head) of last-stage larvae of the black swallowtail butterfly (Papilio polyxenes Fabr.) and the fall armyworm [Spodoptera frugiperda (J. E. Smith)]. The two substrates were metabolized by 10,000g supernatant microsomal preparations from both species. Evidence gained through the use of a specific inhibitor and cofactor indicated that mixed-function microsomal oxidases were major factors in the metabolism and that the specific activity of this enzyme system was considerably higher in midgut preparations fromP. polyxenes than in similar preparations fromS. frugiperda. Aldrin was metabolized 3–4 times faster byP. polyxenes, and xanthotoxin 6–6.5 times faster.     

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.     

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

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

Evaluation of Synergism in the Feeding Deterrence of Some Furanocoumarins on Spodoptera littoralis

The phagodepression activity of five furanocoumarins (FC), bergapten, xanthotoxin, psoralen, imperatorin, and angelicin, has been studied against larvae of Egyptian cotton leafworm Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) using a leaf-disk choice bioassay. The dose range used was 0–10 g/cm² for linear furocoumarins and 0–30 g/cm² for angelicin, the angular furanocoumarin. Dose–feeding deterrency activity correlations were governed by various sigmoidal functions, except in the case of imperatorin and bergapten, which had dose–response curves showing irregular traces with two maxima. All five FCs induced various degrees of phagodepression in S. littoralis; at 3 g/cm², the relative feeding deterrence was bergapten = xanthotoxin > psoralen = imperatorin = angelicin. Structure–activity correlations indicated that a methoxy group on C-5 or C-8 enhanced the activity. Comparison of experimental feeding deterrence of binary mixtures of imperatorin with xanthotoxin, bergapten, or psoralen at various relative concentrations with the calculated additive activity of each combination indicated that a proportion of 40% or more of imperatorin may exert a greater antifeedant effect on S. littoralis than the sum of individual compounds. The effect of angelicin also was examined in combination with psoralen, both of which are present in Psoralea plants. Their mixtures yielded a clear synergistic effect in the 20–80% angelicin range (coactivity coefficient = 124–133). By contrast, the effect of angelicin on xanthotoxin was only additive. In the dose–response curves of imperatorin/bergapten combinations, synergism was found at >60% imperatorin relative concentration and 1 g/cm², whereas lower proportions led to antagonism. The threshold between the two opposing effects was found to depend on the total FC concentration employed. Some natural systems contain FCs in the range of synergistic proportions recorded here and, thus, may have been produced by the host to increase its defensive effect at a lower metabolic cost.     

Defense Secretion of Prorhinotermes simplex: Toxicity to Insecticide Susceptible and Resistant House Fly

(E)-1-Nitropentadec-1-ene (NPE), the main component of the defense secretion of Prorhinotermes simplex soldiers, is toxic to both insecticide-susceptible (S) as well as to insecticide-resistant (R) strains of the house fly, Musca domestica. The LD₅₀ is 11.7 g/female fly for the S strain and 9.7 g for the R strain. The same efficacy of NPE on R and S strains indicates a different mechanism of action compared to conventional chlorinated, organophosphorus, and pyrethroid insecticides. Termite nestmates are protected against NPE by a specific detoxification mechanism. 1-Nitropentadecane, the detoxification product of NPE is nearly nontoxic to house flies, and doses up to 160 g/fly caused only very low mortality.     

Induction of cytochrome P450-mediated detoxification of xanthotoxin in the black swallowtail

Xanthotoxin is a phototoxic allomone found in many of the host plants of the black swallowtail,Papilio polyxenes (Lepidoptera: Papilionidae). When added to the diet of final instar larvae, xanthotoxin can induce the cytochrome P450 monooxygenase (P450) activity in midgut microsomes by which it is detoxified. Induction is dose-dependent, increasing sevenfold when larvae feed on parsley treated topically with xanthotoxin at 0.5 or 1.0% fresh weight. Although xanthotoxin exerts much of its toxic effects when photoactivated by ultraviolet light, induction of P450 activity did not differ in the presence or absence of ultraviolet light. Despite a 4.7-fold induction of xanthotoxin-metabolizing P450 activity, total P450 content measured in the same microsomal samples did not increase significantly. These data indicate that multiple forms of P450 exist in the black swallowtail midgut and that they are differentially induced by xanthotoxin.     

Metabolism and excretion of the furanocoumarin xanthotoxin by parsnip webworm,Depressaria pastinacella

The parsnip webworm,Depressaria pastinacella, feeds on plants containing high concentrations of furanocoumarins. compounds toxic to many organisms. Parsnip webworm larvae were fed radiolabeled xanthotoxin to quantify the detoxification of this furanocoumarin. They metabolized approximately 95% of the ingested xanthotoxin, indicating that metabolic detoxification is important in their tolerance to this allelochemical. Excretion of xanthotoxin and its metabolites was not restricted to the frass but also occurred by means of the silk glands. The silk glands contained half as much of the tritiated compounds as the rest of the body. Because of the feeding habits of this insect, such an excretory pathway may have implications for interactions with predators and pathogens.     

Factors influencing distribution ofDiabrotica spp. in blossoms of cultivatedCucurbita spp.

Cultivars representing three species ofCucurbita were examined for blossom preference byDiabrotica spp.C. maxima cultivars were found to be preferred byD. undecimpunctata howardi over those ofC. pepo andC. moschata.D. virgifera virgifera preferredC. maxima and the Connecticut Field cultivar ofC. pepo.C. moschata and other cultivars ofC. pepo were not preferred. Cultivars were examined for differences in floral volatile release, blossom cucurbitacin content, and pollen content of male blossoms.C. maxima male blossoms released a larger quantity of volatiles thanC. pepo orC. moschata. Also, onlyC. maxima male blossoms contained cucurbitacins. Cultivars ofC. moschata contained the largest quantities of pollen, but all three species contained relatively large quantities. The data indicate a correspondence ofD. u. howardi distribution in the field with high volatile release rates and high cucurbitacin levels that are found inC. maxima blossoms.D. v. virgifera distribution appears to be somewhat independent of these factors since this species was abundant in blossoms of aC. pepo cultivar as well as cultivars ofC. maxima.Coleoptera: Chyrsomelidae.     

Impact of UV radiation on activity of linear furanocoumarins andBacillus thuringiensis var.Kurstaki againstSpodoptera exigua: Implications for tritrophic interactions

Acidic fogs with a pH of 2.0 and duration of 2 hr did not reduce the efficacy ofBacillus thuringiensis var.Kurstaki (Berliner). Therefore, the impact of UV radiation was investigated on the interactions between (1) levels of the antibacterial linear furanocoumarins psoralen, bergapten, and xanthotoxin inApium graveolens (L.) occurring following a 2.0 pH acidic fog episode, (2) the noctuidSpodoptera exigua (Hübner), and (3) a sublethal dosage of the microbial pathogenB. thuringiensis var.Kurstaki. Mean time to pupation in the absence of UV radiation (survival was too low to conduct this analysis for insects exposed to UV) was significantly extended by the addition of either psoralens orB. thuringiensis. Larvae developing on diets containingB. thuringiensis plus psoralens required nearly 40% longer to pupate than controls, but their effects were additive as the interaction was not significant. Although the mean times to adult emergence were significantly different, time spent in the pupal stage did not vary significantly between treatments, indicating that increases in larval developmental time were responsible for the observed decrease in developmental rate. Mean time to mortality, a weighted average time of death, was not significantly affected by any of the treatments. In a 2 × 2 × 2 factorial analysis, all main effects (linear furanocoumarins.B. thuringiensis, UV radiation) reduced survival significantly, as did the three-way interaction. Thus, antagonistic interactions with psoralens that would reduce the effectiveness ofB. thuringiensis in the field were not observed. When pairs of main effects were nested within the two levels (presence and absence) of the third factor, several two-way interactions were found. Interestingly, the activity ofB. thuringiensis and the psoralens, individually or in combination, was enhanced by exposure to UV radiation. Implications of this research are discussed for both natural and agricultural ecosystems.Lepidoptera: Noctuidae.     

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.     

Attraction ofCarpophilus spp. (Coleoptera: Nitidulidae) to synthetic aggregation pheromones and host-related coattractants in Australian stone fruit orchards: Beetle phenology and pheromone dose studies

Synthetic aggregation pheromones ofCarpophilus hemipterus (L.) andCarpophilus mutilatus Erichson were field tested during a 10-month period in southern New South Wales stone fruit orchards to determineCarpophilus spp. phenology and the effect of two pheromone doses on attraction. Aggregation pheromones synergize the attraction of host volatiles toCarpophilus spp. Four major species,C. hemipterus, C. mutilatus, C. davidsoni Dobson andC. (Urophorus) humeralis (F.), were trapped, with greater numbers of each species inC. hemipterus pheromone/fermenting whole-wheat breaddough-baited traps, than in dough-only-traps. InC. mutilatus pheromone/ fermenting-dough-baited traps, onlyC. mutilatus andC. davidsoni responded in greater numbers than to dough-only traps. Beetles first appeared in traps in late September (early spring) when daily maximum temperatures averaged 17.5C. Trappings reached a peak during October and declined to very low levels in November–December (late spring-early summer). Numbers trapped of all species increased during February–March (late summer–early autumn), presumably due to the presence of abundant host resources (ripening and fallen fruit), and continued at high levels until May (late autumn). An 18-week study demonstrated significantly greater responses byCarpophilus spp. to 5000-g than to 500-g doses of C.hemipterus andC. mutilatus pheromones. Greatest responses to 5000g were recorded forC. hemipterus andC. mutilatus responding to their own pheromones (increased attraction over dough alone of 259x and 21.2x respectively). Implications of the study and the potential for using synthetic aggregation pheromones for managingCarpophilus spp. populations in Australian stone fruit are discussed.     

Variation in aphid alarm pheromone content among glandular and eglandular-hairedMedicago accessions

Pea (Acyrthosiphon pisum Harris) and blue alfalfa aphid (A. kondoi Shinji) deterrency in alfalfa (Medicago saliva L.) may result from incorporating higher levels of the aphid alarm pheromone,(E)--farnesene relative to(E)--caryophyllene. We evaluated five eglandular and two glandular-haired alfalfa accessions for differences in(E)--farnesene and(E)--caryophyllene content under glasshouse conditions using supercritical fluid extraction and gas chromatography. In addition, pea and blue alfalfa aphid olfactory behavioral tests were conducted uponMedicago species containing different ratios of(E)--famesene relative to(E)--caryophyllene. No differences in(E)--caryophyllene content were observed among the seven entries (=0.42 ng/g plant material). Significant differences (P 0.05) among entries were observed for(E)--famesene content, with KS94GH6 exhibiting the highest (1.18 ng/g), and CUF 101 the lowest levels (0.35 ng/g). Elite tetraploid sources possessed significantly lower levels (=0.42 ng/g) of(E)--farnesene than did wild and cultivated diploid accessions (=0.83 ng/g). Olfactory behavioral tests for both the pea and blue alfalfa aphids demonstrated KS94GH6 repelled aphids while cultivated alfalfa types attracted aphids in each case. Previously demonstrated aphid resistance in diploid KS94GH6 may result from superior(E)-- farnesene levels, but(E)--farnesene is probably not a factor in cultivated alfalfa resistance. Finally, accession KS94GH6 could act as an excellent germplasm resource for the incorporation of higher(E)--farnesene levels into cultivated alfalfa.Research supported by the New Mexico State University Agricultural Experiment Station.     

Glucosinolate levels in cotyledons of mustard,Brassica juncea L. and rape,B. napus L. do not determine feeding rates of flea beetle,Phyllotreta cruciferae (Goeze)

Sinigrin (allyl glucosinolate), the major glucosinolate in the cotyledons ofBrassica juncea cv. Cutlass, occurred in the highest concentration and amount at seedling emergence and declined during growth. Glucobrassicin (3-indolylmethyl glucosinolate), the major glucosinolate in the cotyledons ofB. napus cv. Westar, occurred in the lowest concentration and amount at seedling emergence. The amount of glucobrassicin per cotyledon pair increased about fourfold during 14 days of growth, but its concentration remained relatively unchanged because of dilution by increasing cotyledon biomass. These different glucosinolate profiles indicate a different metabolic control and different biological function for sinigrin and glucobrassicin. The flea beetle,Phyllotreta cruciferae Goeze, does not discriminate between cotyledons having sinigrin or glucobrassicin since the two crucifers were fed upon equally in choice tests. Restricting the concentration of sulfur in the nutrient medium accelerated the decline of sinigrin inB. juncea cv. Cutlass but did not alter the feeding rate ofP. cruciferae compared to controls. Sulfur restriction reduced glucobrassicin inB. napus cv. Westar to undetectable levels and somewhat reduced the feeding rate of P.Cruciferae. Nevertheless,P. cruciferae still fed actively on cotyledons ofB. napus cv. Westar depleted of glucosinolates and severely damaged many of them. Since glucosinolate type and concentration had little effect on feeding response, reduction or elimination of foliar glucosinolates alone would not seem a useful strategy for protecting seedlings of these two crucifers from flea beetle damage.     

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)--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/cm² 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.     

Receptor cells inIps typographus andDendroctonus micans specific to pheromones of the reciprocal genus

Olfactory receptor cells were studied electrophysiologically inIps typographus andDendroctonus micans. The investigation revealed cells which were keyed to pheromone compounds characteristic of the reciprocal genus. Thus, cells keyed toexo-brevicomin were found inI. typographus, whereas cells keyed to (+)-ipsdienol were present inD. micans. Laboratory behavioral tests indicated an attractive effect of the two compounds on beetles of the reciprocal genus. InI. typographus the effect ofexo-brevicomin predominantly concerned males and enhanced their response to the pheromone ipslure. It is suggested thatexo-brevicomin serves as an interspecific attractant forI. typographus, which may be guided by pheromone compounds of the reciprocal genus in finding suitable breeding material. The function of (+)-ipsdienol inD. micans is more uncertain. It may be either a pheromone or an interspecific messenger.     

Terminal steps in pheromone biosynthesis byHeliothis virescens andH. zea

In vivo application to the sex pheromone gland ofHeliothis Virescens andH. Zea of large quantities of alcohols normally present in small amounts resulted in the preferential conversion of the alcohols to the corresponding pheromonal aldehydes. Amounts of the minor component aldehydes were increased up to 15-fold by selectively applying large quantities of the alcohol precursors. Using this technique, we have inducedH. virescens to convert bombykol, the sex pheromone of the silkworm, to the corresponding aldehyde, bombykal, and have induced femaleH. zea to produce the same sex pheromone components used byH. virescens by applying tetradecanol and (Z)-9-tetradecenol to the surface of the gland. Further, treatedH. zea females were attractive toH. virescens males and caused males to attempt interspecific copulation repeatedly. We have also found that the enyzme involved in this conversion is dependent on the presence of molecular oxygen, indicating that a nonspecific alcohol oxidase is responsible for the terminal biosynthetic step in pheromone production by bothH. virescens andH. zea.Mention of a commercial product or proprietary does not constitute endorsement by either the University of Guelph or U.S.D.A.     

Soybean phytoalexin,glyceollin, prevents accumulation of aflatoxin B1 in cultures ofAspergillus flavus

The soybean phytoalexin, glyceollin, suppresses the accumulation of aflatoxin B₁ in cultures ofAspergillus flavus. At concentrations of 6.25g/ ml and 62.5g/ml, glyceollin causes 70% and 95% decreases in the maximum observed levels of aflatoxin B₁, respectively. In contrast to the dramatic effect on aflatoxin B₁ levels, these concentrations have little effect on fungal growth. For example, at 62.5g/ml in liquid culture, glyceollin causes a barely discernible lag in the beginning of growth and a 11.5% decrease in maximum fungal mass. When the same concentration of glyceollin is added to the colony margin on semisolid medium, an inhibition zone is formed and then overgrown in one day. Glyceollin appears to act by inhibiting aflatoxin B₁ synthesis, since the rate of aflatoxin B₁ breakdown is not increased in fungal cultures that have been grown in the presence of glyceollin. Glyceollin does accumulate in viable soybean seeds that have been infected withAspergillus flavus. Such seeds accumulate aflatoxin B₁ at one-third the rate of non-glyceollin-producing, nonviable seeds. These results suggest that the synthesis of glyceollin in infected seeds may explain, at least in part, why aflatoxin contamination of soybeans is not a common problem.     

(E4,E10)-dodecadienyl acetate: Novel sex pheromone component of tentiform leafminer,Phyllonorycter mespilella (Hübner) (Lepidoptera: Gracillariidae)

(E4,E10)-dodecadienyl acetate (E4,E10-12OAc) is a newly discovered sex pheromone component of the tentiform leafminer,Phyllonorycter mespilella (Hübner). In apple orchards, traps baited with 1g ofE4,E1012OAc attractedP. mespilella in British Columbia andP. blancardella (F.) in Massachusetts and Nova Scotia. The compound was identified inP. mespilella by gas chromatographic-electroantennographic analysis (GC-EAD) of pheromone gland extracts, retention index calculations, EAD profiles toE3 toE10 dodecenyl acetates, and synthesis of candidate pheromone components. Even thoughE4,E10-12OAc was not detected in gland extracts by GC-mass spectroscopy, several factors indicate that it is female-produced. Antennal responses to gland extracts coincided with authenticE4,E10-12OAc on four GC columns with different retention characteristics.E4,E10-12OAc andE10-12OAc, a known female-produced pheromone component, elicited equally strong EAD responses. In field tests,E4,E10-12OAc was two to four times more attractive thanE10-12OAc. There was no additive or synergistic effect between the two components.     

Oviposition stimulants and deterrents regulating differential acceptance ofIberis amara byPieris rapae andP. napi oleracea

Iberis amara (Cruciferae) contains both stimulants and deterrents that are involved in regulating oviposition byPieris rapae andP. napi oleracea. The most active deterrents toP. rapae isolated from butanol extracts of the plant were found to be 2-O--d-glucosyl cucurbitacin I and 2-O--d-glucosyl cucurbitacin E. However,P. napi oleracea was behaviorally insensitive to these compounds and was only weakly deterred by other individual fractions of the butanol extract. Stimulant activity of the postbutanol water extract ofI. amara was associated with glucosinolates. The most abundant of these was identified as sinigrin, and a relatively minor component was shown to be glucoiberin. The isolated sinigrin was more stimulatory toP. rapae than was the glucoiberin-containing fraction, butP. napi oleracea was stimulated as strongly by the glucoiberin fraction, even though the concentration of this compound was much lower. The contrasting responses of the twoPieris species to the deterrents and stimulants inI. amara can explain the differential acceptance of the plant by these butterflies.