Hisashi Ômura

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.

Priority of color over scent during flower visitation by adult Vanessa indica butterflies.

Most flower visitors innately prefer a particular color and scent, and use them as cues for flower recognition and selection. However, in most cases, since color and scent serve as a combined signal, not only does the preference for an individual cue, but also the preference hierarchy among different cues, influence their flower visitation. In the present study, we attempted to reveal (1) the chromatic and (2) the olfactory cues that stimulate flower visiting, and (3) the preference hierarchy between these cues, using the naïve adult butterfly Vanessa indica. When we offered 12 different-colored (six chromatic and six achromatic) paper flower models, V. indica showed a color preference for yellow and blue. When we examined the proboscis extension reflex (PER) of V. indica towards 16 individual compounds identified in the floral scents from two nectar plants belonging to the family Compositae, Taraxacum officinale and Cirsium japonicum, six compounds were found to have relatively high PER-eliciting activities, including benzaldehyde, acetophenone, and (E+Z)-nerolidol. When we combined color and scent cues in two-choice bioassays, where butterflies were offered flower models that were purple (a relatively unattractive color), the models scented with these active compounds were significantly more attractive than the odorless controls. In addition, synthetic blends mimicking the floral scents of T. officinale and C. japonicum (at doses equivalent to that of ten flowers) enhanced the number of visits to the scented models. However, the effect of odorizing was not conspicuous in parallel bioassays when yellow flower models were used, and the butterflies also significantly preferred odorless yellow models to scented purple models. These results demonstrate that V. indica depends primarily on color and secondarily on scent during flower visitation.

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.

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.

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.

1-Octen-3-ol Together with Geosmin: New Secretion Compounds from a Polydesmid Millipede, Niponia nodulosa

The small millipede Niponia nodulosa (Polydesmida: Cryptodesmidae) emits an earthy smell when disturbed. This smell was obtained from hexane extracts from both sexes at the sixth and seventh instars and adult stages, and was found by GC/MS analyses to be composed of two compounds, 1-octen-3-ol as the major component and geosmin as the minor component. This is the first report of these compounds in secreted substances from millipedes. As they showed little repellent activity against foraging ants in bioassays, their biological function as defensive allomones was not clearly elucidated. However, both compounds provoked negative chemotaxis of conspecific millipedes in a Y-tube olfactometer, especially 1-octen-3-ol, which was active at a dose equivalent to the content of a single millipede. Such biological activity suggests that this compound is involved in intraspecific communication in this millipede and may play a role as an alarm pheromone.

From Terpenoids to Aliphatic Acids: Further Evidence for Late-Instar Switch in Osmeterial Defense as a Characteristic Trait of Swallowtail Butterflies in the Tribe Papilionini

We compared the chemical compositions of the osmeterial secretions of fourth and fifth (last) instars of eight swallowtail species of the tribe Papilionini. Four species (Papilio demoleus, P. polytes, P. paris, and P. macilentus) are Asian Rutaceae-feeding swallowtails. The other four (Chilasa epicydes, C. agestor, P. troilus, and P. glaucus) represent more distant clades within the Papilionini and species with larval hosts in other plant families. We conducted a quantitative analysis for six species, but only qualitative analysis for P. glaucus and C. agestor. In all eight species, regardless of larval host plant, secretions of the fourth instar principally consisted of mono- and sesquiterpene hydrocarbons, whereas those of the fifth instar comprised aliphatic acids and their esters. Consistent with earlier findings, our results suggest that this “heterogeneous” pattern of osmeterial chemistry, not seen in other tribes, may characterize the Papilionini as a whole. Unlike those of most Papilio species, the fourth and fifth instars of Chilasa species resemble each other in body coloration. Thus, the heterogeneous osmeterial pattern is not necessarily associated with color change in papilionid larvae. The major terpenoids identified in fourth instar larval secretions from the six species were α-pinene, sabinene, β-myrcene, limonene, β-phellandrene, (Z)-β-ocimene, (E)-β-ocimene, p-mentha-1,4(8)-diene, β-elemene, β-caryophyllene, (E)-β-farnesene, (3Z,6E)-α-farnesene, (Z)-α-bisabolene, germacrene-A, (E)-α-bisabolene, and germacrene-B. The profiles for individual species differed both qualitatively and quantitatively from one another, and certain species also secreted methyl 3-hydroxy-n-butyrate and oxygenated sesquiterpenes in relatively large proportions. Secretions from fifth instars were composed of varying proportions of isobutyric, 2-methylbutyric, and acetic acids, and methyl and ethyl (minor) esters of both isobutyric and 2-methylbutyric acids. The heterogeneity of osmeterial chemistry in the tribe Papilionini may represent fine-tuning of chemical defense in response to shifting predation pressures as the larvae age and grow.

Tolerance to fermentation products in sugar reception: gustatory adaptation of adult butterfly proboscis for feeding on rotting foods.

Adult Vanessaindica and Argyreushyperbius frequently forage on flower nectar, but the former also utilizes tree sap and rotting fruits. Compared to flower nectar, these rotting foods are characterized by low sugar concentrations and the presence of fermentation products (ethanol and acetic acid). We suspected that gustatory responses by the receptors on the proboscis might differ in these species. Among the three sugars tested, sucrose elicited the greatest probing (behavioral) responses and was followed by fructose and glucose. A. hyperbius showed higher sugar sensitivity than V. indica in probing responsiveness. In electrophysiological responses of the proboscis sensilla, V. indica was slightly more sensitive than A. hyperbius to glucose and lower concentrations of the other sugars. The sugar reception in A. hyperbius was strongly inhibited by fermentation products, particularly acetic acid at natural concentrations. In contrast, V. indica was noticeably less susceptible to them than A. hyperbius, and its behavioral and sensory responses to sucrose were enhanced by 5–20% (w/v) ethanol. Thus, V. indica not only possesses tolerance to fermentation products but may perceive them as synergists for sugar reception. To utilize rotting foods, such tolerance might be more necessary than high sugar sensitivity.

Divergent behavioral and electrophysiological taste responses in the mid-legs of adult butterflies, Vanessa indica and Argyreus hyperbius.

Adult nymphalid butterflies possess sensilla trichodea (ST) that perceive taste in their walking legs. We examined whether the gustatory responses to mid-leg tarsal stimulation were different between Vanessa indica (rotting-food feeder) and Argyreus hyperbius (flower-nectar feeder). Sucrose, fructose, and glucose elicited behavioral responses (proboscis extension reflex: PER) and electrophysiological responses (spikes) from ST. Sugar responsiveness was similar in both species, where sucrose was the most stimulatory. Two fermentation products, ethanol and acetic acid, never induced PERs but elicited large-amplitude spikes at a concentration of >1% (w/v). The two species significantly differed in responsiveness to the binary mixtures of sucrose and the fermentation products. Ethanol enhanced the sugar responses of V. indica but slightly inhibited those of A. hyperbius. Although acetic acid suppressed the sugar responses of both species, V. indica was less susceptible than A. hyperbius. When concentration of the fermentation products increased, binary mixtures evoked large spikes together with small ones regarded as the sucrose responses. Unlike the proboscal sensilla in our previous study, the tarsal ST of both species unambiguously responded to fermentation products. These results demonstrate that the tarsal gustatory sense of V. indica is adaptive to the use of rotting foods.