Nanao Hayashi

IDENTIFICATION OF FLORAL VOLATILES FROM Ligustrum japonicum THAT STIMULATE FLOWER- VISITING BY CABBAGE BUTTERFLY, Pieris rapae

Floral scent compounds of Ligustrum japonicum that affect the foraging behavior of Pieris rapae adults were examined by means of chemical analyses, electroantennogram (EAG) responses, and behavioral bioassays; the behavioral biossays consisted of two tests: reflex extension of proboscis (REP) in response to odor, and attraction to scented and unscented artificial flowers. More than 30 compounds, including 2-phenylethanol, benzyl alcohol, and methyl phenylacetate as the major components were identified from L. japonicum flowers. Of these, 22 compounds were tested for their effect on foraging behavior. Phenylacetaldehyde (PA), 2-phenylethanol (PE), and 6-methylhept-5-en-2-one (MHO) elicited the highest REP responses, and benzaldehyde (BA) and methyl phenylacetate (MPA) evoked intermediate REP responses. EAG responses were not necessarily correlated with REP activities; the three high-REP compounds gave only moderate EAG responses, whereas two other compounds (ethyl phenylacetate and 2-phenylethyl acetate) that released high EAG responses showed low REP activities. In two-choice behavioral bioassays, flower models scented with any one of these high-REP compounds attracted significantly more adults, while compounds with low REP activities exhibited weak or no appreciable attractiveness. This suggests that the REP responsiveness closely reflects the attractiveness of a compound and could be an effective measure in elucidating which chemical attractants are involved in flower-visiting. A synthetic blend of five floral chemicals (PA, PE, MHO, BA, and MPA) displayed an attractiveness that was comparable to that of the floral extract and was more effective in attractiveness than the compounds tested singly. Consequently, it is highly likely that the flower-visiting by P. rapae to L. japonicum is mediated largely by floral scent chemicals and that a synergistic effect of the five floral components would be most responsible for attraction of the butterfly to this flower. The present results also strongly suggest that specific floral volatiles may facilitate close-range flower location by P. rapae, could serve in part as a cue for recognizing food sources, and also be profoundly implicated in flower preference.

Chemical and Chromatic Bases for Preferential Visiting By the Cabbage Butterfly, Pieris rapae, to Rape Flowers

Scent and coloration of corolla were examined as floral attributes responsible for preferential visiting by the cabbage butterfly, Pieris rapae, to rape flower, Brassica rapa. Floral volatile components that release the flower-visiting behavior of the butterfly were identified by chemical analyses, electroantennography (EAG), and two behavioral bioassays: proboscis extension reflex (PER) in response to odor and attraction to artificial flowers. GC and GC-MS analyses of the headspace volatiles from the flowers revealed the presence of six aromatic compounds, benzaldehyde, phenylacetaldehyde, benzyl alcohol, 2-phenylethanol, phenylacetonitrile, and indole in decreasing order of quantity. Of these, phenylacetaldehyde elicited the highest response in the PER assay. While benzyl alcohol, 2-phenylethanol, benzaldehyde, and phenylacetonitrile evoked moderate responses, the PER-eliciting activity of indole was very weak. In two-choice behavioral bioassays, artificial flowers scented with any one of these PER-active compounds attracted significantly more butterflies than control (unscented) flowers, whereas those treated with indole were almost inactive. The EAG activities of the six chemicals were not high and were about the same at a low dose (1 μg), but phenylacetaldehyde elicited a much stronger response from both sexes at higher doses (10 and 100 μg). An overall profile of EAG responses at a dose of 100 μg was analogous to that of PER performance, suggesting that floral volatiles may be involved in close-range location or recognition of flowers rather than long-range attraction. By spectroscopic and UV-photographic examinations of rape flower, the central part of the corolla was found to absorb UV rays in marked contrast to the other parts, which reflected near-UV rays (λmax = 350 nm). This indicates that the flower is endowed with a conspicuous nectar guide that is probably an important visual stimulus for attracting foraging adults of P. rapae. Consequently, the present findings strongly suggest that this elaborate pollination strategy of rape flower, characterized by its good combination of olfactory and visual attractiveness, accounts for preferential visiting by the cabbage butterfly to the flower.

Male sex pheromone of a giant danaine butterfly,Idea leuconoe

Males of a giant danaine butterfly,Idea leuconoe, display hairpencils during courtship. The females were visually attracted to and olfactorily arrested by an artificial butterfly model to which male hairpencil extracts were added. The hairpencil extracts contained a complex mixture of volatiles, including pyrrolizidine alkaloid (PA) derivatives (danaidone, viridifloric β-lactone), aromatics (phenol,p-cresol, benzoic acid), terpenoids (geranyl methyl thioether, (E,E)-farnesol), a series of γ-lactones (6-hydroxy-4-undecanolides and its homologs), hydrocarbons [(Z)-9-tricosene, etc.], and several compounds with higher molecular weight. A mixture of the major volatiles applied to a butterfly dummy strongly elicited an abdomen-curling acceptance posture in females. Viridifloric β-lactone and danaidone induced significant electroantennogram responses on the females antennae, suggesting their principal role together with other hairpencil components as a sex pheromone to seduce females.I. leuconoe males seem to acquire the precursor for both of the PA fragments from the host plant,Parsonsia laevigata (Apocynaceae), during the larval stage; thereby they do not show pharmacophagous behavior towards PA-containing plants during the adult stage. However, males are pharmacophagously attracted to and feed on a number of simple phenolic compounds in a manner similar to other danaine species towards PAs. Wild males sequester one of the phagostimulants, (−)-mellein, in the hairpencils in varying quantities. Phenolic compounds incorporated in the hairpencils may act primarily as warning odors linked with the defensive PAs present in the body tissues.

Floral Scent of Osmanthus fragrans Discourages Foraging Behavior of Cabbage Butterfly, Pieris rapae

Field observations revealed that flowers of Osmanthus fragrans attract few insect species despite their strong scent and vivid coloration (yellowish orange). Floral volatiles of the plant were thus examined for suspected repellency to Pieris rapae, a potential visitor that never visits this flower. The influence of odor components on the foraging behavior of adult butterflies was assessed by two behavioral bioassays: proboscis extension reflex (PER) in response to olfactory stimuli and the frequency of visits to artificial flowers. In the PER bioassays, a binary mixture composed of a test material and 2-phenylethanol (a positive standard compound) was used, and the negative effect of test materials was appraised based on the degree of suppression caused compared with the original PER performance evoked by 2-phenylethanol alone. Isopentane extract of the flower exhibited significant deterrency in the PER test and remarkable repellency in the flower-visiting experiment, thereby suggesting the presence of a repellent(s) in the floral volatiles. Fractionation of the extract by silica gel column chromatography yielded an active fraction (based on PER performance), which, upon GC and GC-MS analyses, was found to consist of γ-decalactone, β-ionone, and linalool oxide (LO) isomers (furanosides and pyranosides) as major components together with small amounts of linalool and α-ionone. γ-Decalactone was strongly deterrent in the PER test, and β-ionone and all LO isomers were weakly deterrent, while the other compounds exhibited no deterrency. In the flower-visiting tests, γ-decalactone showed notable repellency, whereas β-ionone had no repellent effect. Among the LO isomers, at least furanosides (cis/trans mixture) and cis-pyranoside were found to be weakly repellent. On the other hand, the butterfly showed strong to weak EAG responses to LO furanosides, cis-LO pyranoside, γ-decalactone, and β-ionone in decreasing order of intensity. The present results clearly indicate that the repellency of a compound is correlated with its deterrent effect on PER but not necessarily with antennal sensitivity.

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.

Pyrrolizidine alkaloids mediate host-plant recognition by ovipositing females of an old world danaid butterfly, Idea leuconoe

A giant danaid butterfly, Idea leuconoe, specializes on apocynaceous plants such as Parsonsia laevigata, which has been reported to contain pyrrolizidine alkaloids. Females of I. leuconoe deposited eggs in response to methanolic extract of P. laevigata, and subsequent bioassay-guided fractionation of the extract revealed that phytochemicals crucial for host recognition by ovipositing females are Parsonsia-specific macrocyclic pyrrolizidine alkaloids including parsonsianine, parsonsianidine, and 17-methylparsonsianidine. Parsonine, another P. laevigata pyrrolizidine component with a keto-dihydropyrrolizine moiety that is closely related in structure to male pheromones of the butterfly, and several nonhost pyrrolizidine alkaloids were entirely inactive. We interpret these data as strong evidence for an ancestral association through herbivory between danaid butterflies and pyrrolizidine alkaloids.

Identification of feeding attractants in oak sap for adults of two nymphalid butterflies, Kaniska canace and Vanessa indica

The active compounds of oak‐sap odour in attracting adults of two butterflies, Kaniska canace (L.) and Vanessa indica (Herbst) (Lepidoptera: Nymphalidae), were identified by chemical analyses, electroantennogram (EAG) and two behavioural assays: proboscis extension reflex (PER) and attraction to artificial tree models. Fourteen compounds were identified from two sap samples collected in 1997 and 1998, of which the major volatiles were ethanol and acetic acid (≈ 900 p.p.m. and 500 p.p.m. in sap, respectively). However, the chemical composition of the minor volatiles varied considerably between the two samples. Among 13 chemicals tested, V. indica showed strong PER to five aliphatic acids (acetic, propionic, butyric, isobutyric and isovaleric), 2‐methylpropan‐1‐ol and 3‐hydroxybutan‐2‐one, whereas the PER‐active compounds for K. canace were these seven compounds and also ethanol, 3‐methylbutan‐1‐ol and 1‐hydroxypropan‐2‐one. In two‐choice behavioural bioassays, the model scented with a sap‐odour mimic, which was an aqueous mixture of the PER‐active compounds, was more attractive to the two butterflies than an unscented control. These results demonstrated that the sap odour stimulates foraging behaviour of the butterfly. Although EAG responses of both butterflies to 3‐methylbutan‐1‐ol and that of V. indica to 2‐methylpropan‐1‐ol were positively dose‐dependent, responses to other compounds were not strong and not dose‐dependent at 1–100 μg doses. These EAG responsiveness suggests that the olfactory receptors for these compounds might be few in the antenna and that the butterflies have enough olfactory sensitivity to the dose of 1 μg.

Phenanthroindolizidine alkaloids from Tylophora tanakae

Abstract From the fresh leaves of Tylophora tanakae , ten phenanthroindolizine alkaloids were isolated. Among them, eight were new alkaloids and their structures were determined. Two known alkaloids were identified as isotylocrebrine and tylophorine.

Components of the ant secretions.

Abstract Studies on the chemistry of the secretions from various ant species have shown that the ants can be classified into four groups on the basis of the major component(s) in their secretions: group A, contains paraffins along with formic acid; group B, contains terpenes; group c, contains considerable amount of paraffins, terpenes and acyclic ketones; and group D, contains aromatic compounds.

Alkaloidal oviposition stimulants for a danaid butterfly,Ideopsis similis L., from a host plant,Tylophora tanakae (Asclepiadaceae)

Chemicals releasing oviposition by an Asclepiadaceae feeder,Ideopsis similis, were identified from a host plant,Tylophora tanakae. A strong positive response was evoked by a methanolic extract of the plant, which proved to contain multiple stimulants. A mixture of two phenanthroindolizidine alkaloids, (+)-isotylocrebrine and (−)-7-demethyltylophorine, isolated from organic fractions, elicited significant ovipositional responses from females.