Chemical constituents of an unacceptable crucifer,Erysimum cheiranthoides,deter feeding byPieris rapae

The wild cruciferErysimum cheiranthoides was found to contain extractable constituents that deterred feeding by larvae of the crucifer specialistPieris rapae when applied to cabbage leaf disks in both choice and nochoice bioassays. High-performance liquid chromatography was used to separate the extract into several fractions, two of which retained the feeding deterrent activity of the extract. UV-absorption spectra of the fractions suggested that one contained cardenolides similar or identical to those reported to deter oviposition byP. rapae onE. cheiranthoides. The other active fraction evidently contains a compound that deters larval feeding but not adult oviposition. The results suggest that the chemical defense ofE. cheiranthoides depends on two types of compounds acting on separate developmental stages of the insect.     

A chemical basis for differential acceptance ofErysimum cheiranthoides by twoPieris species

Wormseed mustard,Erysimum cheiranthoides, is unacceptable as a host for the cabbage butterfly,Pieris rapae. However, it is preferred for oviposition byPieris napi oleracea in the greenhouse. Isolation and identification of the oviposition stimulants toP. napi oleracea were accomplished by C₁₈ open-column chromatography, TLC, ion-exchange chromatography, HPLC, UV, and NMR spectroscopy. Glucoiberin and glucocheirolin were identified as the most active stimulants. The extracted glucoiberin was as stimulatory as glucocheirolin, although its concentration in theErysimum plants was about 10 times lower than that of glucocheirolin. These glucosinolates were only weak stimulants toP. rapae. Furthermore,P. rapae was strongly deterred by the cardenolides, erysimoside and erychroside, fromE. cheiranthoides, andP. napi oleracea was less sensitive to these compounds. No other deterrent toP. napi oleracea was detected in this plant species. The results explain the differential acceptance ofE. cheiranthoides by these twoPieris species.     

Oviposition stimulants for the tropical swallowtail butterfly,Papilio polytes,feeding on a rutaceous plant,Toddalia asiatica

In nature, Papilio polytes utilizes a limited range of rutaceous plants as hosts. We isolated and identified oviposition stimulants for the butterfly from the foliage of its primary host plant Toddalia asiatica. Females readily deposited eggs in response to a methanolic extract of the plant. Partition of the extract with organic solvents revealed that chemicals responsible for eliciting egg-laying resided in a water-soluble fraction. Further bioassay-guided fractionation of the active fraction by column chromatography, preparative TLC, and HPLC led to the isolation of two oviposition stimulants. One was isolated from an amphoteric fraction and identified as trans-4-hydroxy-N-methyl-L-proline [(–)-(2S,4R)-4-hydroxy-1-methyl pyrrolidine-2-carboxylic acid; HMP]. The other, isolated from an acidic fraction, was identified as 2-C-methyl-D-erythronic acid [(–)-(2R,3R)-2-methyl-2,3,4-trihydroxybutanoic acid; MEA]. HMP alone evoked significant oviposition-stimulatory activity, although this was much lower than that of the original water-soluble fraction. MEA, on the other hand, alone did not elicit positive responses from females. However, HMP, when assayed in combination with MEA, markedly enhanced the female response, and the mixture was as active in stimulating oviposition as were the original water-soluble fraction and the plant foliage. We conclude that HMP is a substance crucial for host recognition by females, while MEA is a synergistic stimulant involved in host recognition and/or preference.     

An Oviposition Stimulant for Spicebush Swallowtail Butterfly, Papilio troilus, from Leaves of Sassafras albidum

Female butterflies of the spicebush swallowtail, Papilio troilus, are specialists, ovipositing on plants in the family Lauraceae. Column chromatography and HPLC were used to isolate an oviposition stimulant from the leaves of one of its hosts, Sassafras albidum. The stimulant was identified as 3-trans-caffeoyl-muco-quinic acid on the basis of FAB-MS and ¹H NMR spectra as compared to a compound previously isolated from another plant. It was not active alone, but it increased the oviposition activity of butterflies when combined with other stimulant(s) at a concentration of 7 ng/mm² leaf surface area. Other caffeoylquinic acid isomers tested did not have this effect. This is the first report of a swallowtail contact oviposition stimulant from a plant in the family Lauraceae.     

Cardenolides as oviposition deterrents to twoPieris species: Structure-activity relationships

Oviposition responses ofPieris rapae andP. napi oleracea to 18 cardenolides were compared under the same conditions. Effects of different concentrations of selected cardenolides were also tested. Most of the compounds were deterrent to oviposition by both insects, but to significantly different degrees.P. rapae were strongly deterred by K-strophanthoside, K-strophanthin-, cymarin, convallatoxin, oleandrin, erysimoside, erychroside, and gitoxigenin. The most deterrent compounds forP. napi oleracea were erychroside, cymarin, erysimoside, convallatoxin, and K-strophanthoside. Strophanthidin-based glycosides were more deterrent than digitoxigenin-based ones, and the number and type of sugar substitutions can have profound effects on activity. Both similarities and contrasts were found in responses ofP. rapae andP. napi oleracea to these cardenolides. Cymarin was equally deterrent to bothPieris species at all concentrations tested. However, when compared withP. rapae, P. napi oleracea was less sensitive to most of the cardenolides.P. napi oleracea was insensitive to K-strophanthin- and oleandrin at 0.5 × 10^–4 M, which were highly deterrent toP. rapae.     

Specialist Weevil, Rhyssomatus lineaticollis, Does Not Spatially Avoid Cardenolide Defenses of Common Milkweed by Ovipositing into Pith Tissue

Rhyssomatus lineaticollis is a milkweed specialist whose larvae feed upon pith parenchyma in ramet stems of the common milkweed, Asclepias syriaca. Compared with other specialist insect herbivores on milkweeds, this curculionid beetle is unusual in that it is cryptically colored and does not sequester cardenolides characteristic of milkweed chemical defense. Based upon optimal defense theory, we predicted that pith tissue would be low in defensive compounds and that oviposition into the pith would spatially avoid cardenolides. We rejected this hypothesis because we found that pith tissue has a relatively high cardenolide concentration compared to cortex, epidermis, and leaf tissues. Moreover, we found total plant cardenolide concentration was lower in plants that contained the beetle eggs. Cardenolide concentrations were different among tissues in intact stems without the pith herbivore compared to stems where it was present. Furthermore, the overall polarity of the cardenolides present varied among plant tissues and between plants with and without R. lineaticollis eggs. Although we found lower concentrations of cardenolide in piths where the eggs were present, the cardenolides present in the pith contained more nonpolar forms, indicating that the plant may be responding to herbivory by increasing toxic efficacy of cardenolide defenses while lowering the total concentration. We suggest that preoviposition behavior by female beetles, which includes feeding on new leaves of the plant, is a mechanism by which females manipulate plant chemistry and assess quantitative and qualitative changes in cardenolide chemistry in response to herbivory prior to oviposition.     

Identification and synthesis of a kairomone inducing oviposition by parasitoidAphytis melinus from California red scale covers

The parasitoid waspAphytis melinus uses a kairomone from the cover of its scale host, California red scale (Aonidiella aurantii), as an oviposition stimulant. The kairomone was isolated from extracts of scale covers, and identified asO-caffeoyltyrosine by a combination of spectroscopic methods. The kairomone was synthesized, and the synthetic compound was determined to be as active as the chemical isolated from scale covers.     

Production of cardiac glycosides by chrysomelid beetles and larvae

Cardenolides were looked for in 17 chrysomelid beetles belonging to 11 genera from three subfamilies, and they were found only inChrysolina andChrysochloa species (Chrysomelinae, Chrysolinini). The food plants of these insects are not known to produce cardenolides. TheChrysochloa and mostChrysolina species secrete a complex mixture of cardenolides, butChrysolina didymata secretes a single compound, andChrysolina carnifex, none. Several quantitative and perhaps qualitative differences were observed in the patterns of cardenolides produced by far distant populations of bothChrysolina polita andC. herbacea, collected in either France and Belgium, or Greece. These differences remain constant from one generation to the other, whatever the food plant is, and appear to be genetic. InC. polita from Greece, the pattern is unchanged after four generations bred in the laboratory onMentha ×villosa, which is known to be without cardenolides. In adults, the cardenolides are released with the secretion of the pronotal and elytral defensive glands, but in the larvae which lack the defensive glands, cardenolides are also produced. The total amount of cardenolides and the complexity of their mixture increases through the life cycle of the insects. The six main cardenolides secreted byC. coerulans were identified as: sarmentogenin, periplogenin, bipindogenin, and their corresponding xylosides.C. didymata secretes only sarmentogenin.     

Isolation and identification of oviposition deterrents to cabbage butterfly,Pieris rapae,fromErysimum cheiranthoides

Avoidance of some crucifer species by the crucifer specialist,Pieris rapae, has been attributed to the presence of oviposition deterrents in these plants. Studies on one such unacceptable plant,Erysimum cheiranthoides, have resulted in the isolation ofn-butanol-soluble deterrents from the alcoholic extract of foliage. The active fraction contained three cardiac glycosides, which were isolated by reversed-phase HPLC and by open column chromatography on silica gel. Chemical and spectral evidence (UV, [¹H]NMR, and FAB-MS) led to the characterization of these compounds as erysimoside (1), erychroside (2), and erycordin (3). Erysimoside and erychroside were strongly deterrent toPieris rapae, but erycordin was inactive. Both active compounds have the same aglycone, strophanthidin (5) and the inner sugar in both cases is a 2,6-dideoxy hexose to which the outer sugar is attached at position C-4. These structural features, which are absent in the inactive compound (3), may represent specific requirements for oviposition deterrent activity.     

Cardenolides fromErysimum cheiranthoides: Feeding deterrents toPieris rapae larvae

Larvae of the cabbage butterfly,Pieris rapae, refuse to feed on the wild mustard,Erysimum cheiranthoides, due to the presence of alcoholextractable deterrents. The active components were extracted inton-BuOH, and this extract was separated into four fractions (I–IV) by reverse-phase HPLC. Fractions III and IV retained the feeding deterrent activity. The activity of fraction III was found to be due to the cardenolide diglycosides 1 and 2, which were previously reported as oviposition deterrents for gravidP. rapae butterflies. Three active compounds were isolated from fraction IV by column chromatography on silica gel followed by reverse-phase HPLC. These compounds were identified as a monoglycoside, digitoxigenin 3-O--D-glucoside (4), and two diglycosides, glucodigigulomethyloside (5) and glucodigifucoside (6). An additional cardenolide isolated from fraction II was identified as cheirotoxin (7). All compounds were identified by UV, NMR (¹H and¹³C), and mass spectrometry, as well as hydrolysis experiments. The feeding deterrent activity of these compounds was compared with that of related commercially available chemicals and other compounds isolated fromE. cheiranthoides.     

Isolation of Three Diterpenoid Acids from Sunflowers,as Oviposition Stimulants for the Banded Sunflower Moth, <Emphasis Type="Italic">Cochylis hospes</Emphasis>

The banded sunflower moth (BSFM), Cochylis hospes Walshingham (Lepidoptera: Cochylidae) is a specialist insect, the larvae of which feed on sunflowers, Helianthus spp., and a few other species of Compositae. It is one of the most important pests of sunflower in the USA. Previous work on H. annuus, the cultivated sunflower, revealed two diterpenoids that function as oviposition stimulants for female BSFM, and that other, more polar compounds also stimulated oviposition. Using a bioassay-guided approach, we isolated three additional diterpenoids, grandifloric acid (1), 15β-hydroxy-ent-trachyloban-19-oic acid (2), and 17-hydroxy-16α-ent-kauran-19-oic acid (3), from polar fractions of pre-bloom sunflower head extracts. In laboratory bioassays, purified natural samples of each of these compounds stimulated oviposition by female BSFM. Structure–activity relationships of the five diterpenoids known to stimulate oviposition by female BSFM are discussed.     

Epicuticular Wax Chemicals in Zea mays Influence Oviposition in Ostrinia nubilalis

The chemical basis of oviposition elicitation in a generalist herbivore was determined by examination of oviposition responses in Ostrinia nubilalis to corn (Zea mays) chemicals in two-choice laboratory bioassays. A pentane extract of corn leaves stimulated oviposition and the activity persisted for three days, indicating that oviposition in O. nubilalis is elicited by low-volatility chemicals. Chemicals in the extract were fractionated by column chromatography on Florisil, using a sequence of solvents of increasing polarity. Bioassays of Florisil fractions indicated that the stimulants were eluted with nonpolar solvents. Positive bioassay results with an extract prepared by dipping corn leaves in pentane for 20 sec for extraction of leaf surface chemicals suggested that some of the active material was present in the leaf epicuticle. Gas chromatographic analyses and comparisons with retention times of standards suggested the presence of several n-alkanes in the dip extract. Five n-alkanes—hexacosane, heptacosane, octacosane, nonacosane, and tritriacontane—known to be present in the epicuticle of corn leaves were bioassayed, and all five elicited oviposition responses. These results suggest that oviposition elicitation in O. nubilalis is influenced by the presence of n-alkanes in the host plant epicuticle.     

Oviposition repellents of mosquitoes: Negative responses elicited by lower aliphatic carboxylic acids

Organic infusions have been shown to elicit discriminatory responses in ovipositing mosquitoes. Previously, we found that a Purina® Lab Chow infusion induces negative oviposition behavior inCulex pipiens quinquefasciatus Say andCulex tarsalis Coquillett. Six aliphatic carboxylic acids isolated from the active fraction of this infusion were acetic, propionic, isobutyric, butyric, isovaleric, and caproic. In the present studies, we have quantified the negative oviposition responses ofCulex mosquitoes to these carboxylic acids in a laboratory bioassay system and have also tested the main acid component, butyric acid, againstCuliseta, Aedes, andAnopheles mosquitoes.     

Attractancy and species specificity of 6-acetoxy-5-hexadecanolide,a mosquito oviposition attractant pheromone

Four stereoisomers of 6-acetoxy-5-hexadecanolide, a mosquito oviposition attractant pheromone, were bioassayed for their activity on mosquitoes. Only (–)-(5R,6S) isomer was active in attractingCulex quinquefasciatus Say females for oviposition at dosages of 0.5 g/100 ml water and above with the floating-cap method. The activity of this isomer increased 50-fold when it was applied directly to the water surface. The other three isomers, (+)-(5S,6R), (+)-(5R,6R), and (–)-(5s, 6s), were not active. The active isomer was ovipositionally attractive not only toC. quinquefasciatus but also toC. tarsalis Coquillett; however, it was 100 times more active in the former than in the latter species.Aedes aegypti (L.) andAnopheles quadrimaculatus Say were not attracted to the pheromone, thus suggesting that the pheromone is genus-specific.     

Identification and bioassay of kairomones forHelicoverpa zea

Hexane extracts of leaves of 307 accessions from 73 host plant species ofHelicoverpa zea were analyzed by gas chromatography (GC) and used forH. zea oviposition and neonate larvae orientation bioassays. The gas chromatographic (GC) retention times of compounds statistically associated with behavioral activity were identified by correlation of GC peak area with oviposition and larval orientation preferences. Although taxonomically diverse in their origin, compounds for study were purified from extracts of species of the genusLycopersicon, due to their relative abundance. The structures of eight long-chain alkanes associated with oviposition preference were assigned by mass spectrometry, and the structures of five similarly associated organic acids and a terpenoid alkene were identified by¹H and¹³C nuclear magnetic resonance spectroscopy. The structures of a number of other phytochemicals from the plant leaves were identified for comparative purposes, including a previously unknown terpene, 7-epizingiberene. Bioassays were performed on the isolated acids and on the alkane wax fractions of severalLycopersicon species, and significant differences were found in oviposition stimulation for both classes of compounds. Of the hundreds of compounds found in the extracts, none were observed to act as oviposition deterrents. The results of these bioassays may be useful in explaining the broad host range ofH. zea, as well as the process and evolution of host plant selection for oviposition.     

Host-Plant Chemistry Influences Oviposition Choice of the Spicebush Swallowtail Butterfly

The spicebush swallowtail, Papilio troilus (L.), lays its eggs on plants in the family Lauraceae. Sassafras [Sassafras albidum (Nutt.) Nees], spicebush [Lindera benzoin (L.) Spreng.], redbay (Persea borbonia (L.)] and camphortree [Cinnamomum camphora (Nees) Eberm.] are four of its known host plants. In one-choice tests, free-flying spicebush swallowtail females laid eggs on chemical extracts of the leaves of each of these four hosts. In two-choice experiments, females always preferred to oviposit on an extract of sassafras compared to extracts of the other three hosts. It was shown for spicebush extract that this response was not due to oviposition experience. Previously we had identified one of the host plant chemicals acting as an oviposition stimulant in sassafras extract as 3-caffeoyl-muco-quinic acid (3-CmQA). Extracts of the other three hosts did not contain this compound. The addition of 3-CmQA alone to spicebush extract did not increase oviposition activity. It did, however, increase discrimination between hosts and nonhosts. When a fraction of sassafras extract containing 3-CmQA and other synergistic stimulants was added to spicebush extract, preference for sassafras extract was no longer recorded. These results show existing differences in oviposition chemistry among host plants of the spicebush swallowtail and how these differences can influence oviposition choice in bioassay experiments.     

Influence of trichome exudates from species ofLycopersicon on oviposition behavior ofHeliothis zea (Boddie)

Cage experiments revealed that accessions of the wild tomato speciesLycopersicon hirsutum were preferred sites for oviposition byHeliothis zea. Hexane extracts from the leaves ofL. hirsutum were also preferred sites of oviposition in choice experiments among extracts from severalLycopersicon species. Extracts ofL. hirsutum were still biologically active several days after application, indicating that the phytochemical(s) involved are relatively stable and of low volatility. Gas Chromatographic analysis of leaf hexane extracts from 12 different accessions of theL. hirsutum complex and three tomato cultivars revealed substantial qualitative and quantitative variation in the chemical composition of these extracts. Comparison of these results with extract oviposition studies implicate a group of structurally related compounds as the active agents. Mass spectroscopy has tentatively identified these compounds as sesquiterpenes with the chemical formula C₁₅H₂₂O₂. These compounds are apparently synthesized and secreted from glandular trichomes on the leaf surface. These phytochemicals did not stimulate ovipositional behavior in females of the cabbage looper,Trichoplusia ni. The existence of genetic variation for the presence and amount of kairomones that serve as cues for insect orientation and oviposition could be utilized in a breeding program to develop tomato cultivars with genetically modified allelochemic profiles that would disrupt the sequential behavioral processes of insect host-plant selection.     

Selective sequestration of milkweed (Asclepias sp.) cardenolides inOncopeltus fasciatus (Dallas) (Hemiptera: Lygaeidae)

The cardenolide content of the gut, wings, and fat body ofOncopeltus fasciatus was examined. The female fat body contained 4–5% of the total cardenolide content of the insect. The cardenolide content of male fat body, and gut and wings of both sexes was below the detection limit of the cardenolide assay. Thin-layer chromatography was used to determine the cardenolide array of various tissues and secretions ofO. fasciatus reared on seeds of a single species of milkweed (A. Speciosa) and adult extracts and dorsolateral space fluid ofO. fasciatus reared on seeds of two species of milkweed with different cardenolide arrays (A. speciosa andA. syriaca). Our results indicate that cardenolides are not sequestered in the insect simply on the basis of polarity and that metabolism and differential excretion of cardenolides are involved in the sequestration of cardenolides inO. fasciatus. The similarities in the cardenolide profiles ofO. fasciatus reared on different food sources, and tissues ofO. fasciatus reared on a single food source indicates that there is regulation of the cardenolide array inO. Fasciatus.     

Response ofNp mutant of pea (Pisum sativum L.) to pea weevil (Bruchus pisorum L.) oviposition and extracts

TheNp mutant of pea (Pisum sativum L.) is characterized by two physiological responses: growth of callus under pea weevil (Bruchus pisorum L., Coleoptera: Bruchidae) oviposition on pods, and formation of neoplastic callus on pods of indoor-grown plants. Although these two responses are conditioned byNp, they are anatomically and physiologically distinguishable, based on sites of origin, distribution pattern, and sensitivity to plant hormones. Further characterization of the response to extracts of pea weevil showed that response of excised pods, measured by callus formation, was log-linear, and treatment with as little as 10^–4 weevil equivalents produced a detectable response. Mated and unmated females contained similar amounts of callus-inducing compound(s), and immature females contained significantly less of the compound(s). Female vetch bruchids (Bruchus brachialis F., Coleoptera: Bruchidae), a related species, contained callus-inducing compound(s), but usually less than pea weevils on a per weevil basis. Males of both species contained less than 10% of the activity of the mature females. Extracts of female black vine weevils, a nonbruchid species, did not stimulate callus formation. Based on partitioning and TLC analysis, the biologically active constitutent(s) was stable and nonpolar. Thus, theNp allele probably conditions sensitivity to a nonpolar component of pea weevil oviposition as a mechanism of resistance to the weevil.     

Plant response to eggs vs. Host marking pheromone as factors inhibiting oviposition byPieris brassicae

Pieris brassicae L. butterflies secrete miriamides onto their eggs. These avenanthramide alkaloids are strong oviposition deterrents when sprayed onto a cabbage leaf. However, these compounds could not be detected in cabbage leaves from which egg batches had been removed two days after deposition and that still showed oviposition deterrency. It was concluded that the miriamides were not directly responsible for the avoidance by females of occupied leaves while searching for an oviposition site. Evidence was obtained that cabbage leaves themselves produce oviposition deterrents in response to egg batches. Fractions containing potent oviposition deterrents could be isolated from surface extracts of leaves from which previously laid egg batches had been removed. The term host marking pheromone that was used previously is not applicable in this case.