Appraisal of the Acceptability of Subtropical Rutaceous Plants for a Swallowtail Butterﬂy, Papilio Protenor Demetrius (Lepidoptera: Papilionidae)

Appl. Entomol. Zool. 42 (1): 121–128 (2007) http://odokon.org/

Appraisal of the acceptability of subtropical rutaceous plants for a swallowtail butterﬂy, Papilio protenor demetrius (Lepidoptera: Papilionidae)

Mamoru CHACHIN, Keiichi HONDA* and Hisashi ÔMURA Department of Biofunctional Science and Technology, Graduate School of Biosphere Science, Hiroshima University; Higashi- hiroshima 739–8528, Japan (Received 18 August 2006; Accepted 20 October 2006)

Abstract A Rutaceae-feeding swallowtail butterfly, Papilio protenor demetrius, exploits only a few plant species as hosts in the field. We examined in detail the acceptability of five potential rutaceous hosts occurring in the subtropics for ovipositing females of a Hiroshima population of the butterfly. The plants tested were Citrus depressa, Toddalia asiatica, Evo- dia meliifolia, Melicope triphylla, and Murraya paniculata, which are distributed mainly in the Southwestern Islands of Japan, thus in allopatry with the butterfly. Female responses to the foliage, methanol extracts and partitioned fractions from these plants were assayed for the presence of oviposition stimulants and/or deterrents. The foliage of C. depressa and T. asiatica strongly stimulated egg-laying, whereas ovipositing females only marginally accepted E. meliifolia and Me. triphylla, and virtually rejected Mu. paniculata. Further experiments with methanol extracts and fractions derived from the respective plants revealed that both C. depressa and T. asiatica contained potent oviposition stimulant(s) particularly in the aqueous fractions, and that the aqueous fractions of E. meliifolia and Mu. paniculata, despite their poor or little acceptability, contained moderate and weak stimulant(s), respectively. However, certain volatile deterrent(s) seemed to be responsible for the rejection of Mu. paniculata by females. Me. triphylla appeared to have neither appreciable stimulant(s) nor deterrent(s). Larval survival on these plants was also recorded as an estimate of fitness. Larvae performed very well on C. depressa. Unexpectedly, however, larvae did not survive on T. asiatica at all, but successfully grew on E. meliifolia. Larvae altogether failed to grow on both Me. triphylla and Mu. paniculata, suggesting the involvement of antifeedant(s) and/or toxic substance(s) in those plants to which larvae were maladapted.

Key words: Host selection; oviposition; larval performance; Papilio protenor demetrius; Rutaceae

made on the basis of a reciprocal balance of posi- INTRODUCTION tive and negative inputs from the plant which are Most butterfly species are phytophagous and ultimately transmitted to the central nervous sys- usually utilize only a limited range of host plants in tem for further processing (Renwick and Chew, nature. Although the host range of an insect is de- 1994; Honda, 1995). Extensive investigations car- termined by a diversity of ecological, geographical, ried out during the last two decades have revealed physiological, and behavioral factors, the key ele- an array of stimulants and deterrents of plant origin ments underlying the host range determination are responsible for oviposition by butterflies and moths phytochemicals; stimuli evoked, in particular, by (Renwick and Chew, 1994; Honda, 1995, 2005; plant secondary metabolites which are thought to Nishida, 1995; Honda and Nishida, 1999). The have a significant effect on host utilization by her- most crucial phase of host assessment in butterflies bivores (e.g. Städler, 1992). It has been fully estab- is “drumming” on the leaf surface with foretarsi lished that phytophagous lepidopterans can detect endowed with gustatory sensila. The majority of specific plant chemicals, which they use to assess phytochemicals directly associated with host recog- the suitability of potential hosts, and the decision nition and discrimination by ovipositing females to accept a given plant by ovipositing females is are those characterized mostly as non-volatile

*To whom correspondence should be addressed at: E-mail: [email protected] DOI: 10.1303/aez.2007.121

121 122 M. CHACHIN et al. secondary metabolites (Honda, 2005). However, allowed to copulate in an outdoor cage (7 m10 m, volatile compounds from host plants, in certain 3.5 m high). These were derived from laboratory cases, are also believed to stimulate egg-laying be- stock cultures originated from females collected in havior and thus promote egg deposition (Feeny et Higashihiroshima City (Hiroshima Prefecture). al., 1989). Larvae were reared on potted plants of Citrus spp. While papilionid butterflies are known to exploit (mainly C. natsudaidai) or F. ailanthoides at 25°C a wide range of plant families as hosts, most under a photoregime of 16L : 8D. Females were fed species of the genus Papilio exclusively utilize ru- with 15% aqueous sucrose solution once daily taceous plants, showing a differential use of allied throughout the experiments. plants depending on the species (Miller, 1987). De- Extraction and fractionation of plant materi- spite extensive work by many scientists, phyto- als. Young leaves of C. depressa, T. asiatica, E. chemical cues and sensory mechanisms regulating meliifolia, Me. triphylla, and Mu. paniculata col- host selection and host preference in these butter- lected in Okinawa Pref. (0.5–1 kg each) were ex- flies remain poorly understood. For a better under- tracted separately with methanol (4–8 l) at room standing of the physiochemical background of temperature for one month. Subsequent fractiona- present-day host utilization and host range evolu- tion of each extract was carried out according to tion in Papilio butterflies, further attempts are the method of Honda (1990), as outlined below. needed to investigate the chemical basis for differ- Each methanolic extract was concentrated in vacuo ential acceptance of potential host plants. below 50°C, and an aliquot of the whole concen- Papilio protenor is a specialist on the Rutaceae. trate, after being dispersed in water, was succes- P. protenor demetrius, a subspecies distributed sively partitioned with chloroform and isobutanol from the main island (Honshu) to the Amami Is- to give three fractions (one aqueous and two or- lands of Japan, utilizes several Citrus plants and a ganic fractions). The chloroform-soluble fraction few species of other genera (Fagara, Zanthoxylum, (Fr. 1) was stored in chloroform. The isobutanol- and Skimmia) as hosts. Another subspecies, P. pro- soluble fraction (Fr. 2) was evaporated to dryness tenor liukiuensis, inhabits more southerly regions, below 50°C, and the residue was re-dissolved in i.e. Okinawa and Yaeyama Islands, where many ru- methanol. The water-soluble fraction (Fr. 3) was taceous plants such as Murraya paniculata, Evodia dissolved in 50% aqueous methanol. All these frac- meliifolia, Fagara ailanthoides, Toddalia asiatica, tions were stored below 0°C until use. Fractions Citrus depressa, Melicope triphylla, Skimmia prepared from the respective plants will hereafter japonica var. lutchuensis, and Zanthoxylum ni- be abbreviated: Fr. 1 of C. depressa and Fr. 2 of T. tidum grow. We have previously reported on the asiatica, for instance, will be designated as Cd-1 affinity of P. protenor demetrius for four temperate and Ta-2, respectively. Concentrations of test sam- rutaceous plants occurring in Honshu; Citrus ples are all shown in % w/v. An initial extract and unshiu, Fagara ailanthoides, Phellodendron three partitioned fractions (Frs. 1–3) of a given amurense, and Orixa japonica (Honda and concentration prepared from the respective plants Hayashi, 1995a). This study of P. protenor were tested for their stimulatory or deterrent effect demetrius deals with a comparative appraisal of fe- on oviposition. male oviposition responses to and the suitability Bioassay for oviposition response. Behavioral for larval growth of five subtropical rutaceous bioassays were conducted by a method similar to plants, C. depressa, T. asiatica, E. meliifolia, Me. that reported previously (Honda, 1986), employing triphylla, and Mu. paniculata. In Japan, these are a green heart-shaped plastic plate (2–5 cm2) as a distributed in the Southwestern Islands, though E. leaf surrogate. Before testing, females were meliifolia is also found in Kyushu and Shikoku. screened daily to assess their responsiveness, and only those that showed positive responses to the foliage of C. natsudaidai (one of the major host MATERIALS AND METHODS plants of P. protenor demetrius; Honda, 1990) and Insects. Adults of P. protenor demetrius sub- negative responses to water alone (control) were jected to behavioral bioassays were 3- to 10-day- chosen. Appraisal of female responsiveness to each old gravid females, which had been hand-paired or sample was made basically in accordance with the Butterfly Host Selection 123 criteria given in previous papers (Honda et al., 1997; Nakayama et al., 2002): In each trial (sample presentation), the response of an individual was scored as 100% for actual egg-laying or equivalent behavior (trying to bring the ovipositor in contact with the underside of the leaf without egg deposition), 50% for half-curling the abdomen with continuous drumming (this behavior took place in- frequently), and 0% for drumming only with no positive response. Trials were replicated more than three times for each individual and the responses of an individual to a given sample were averaged. For all trials, merely alighting on ovipositional sub- Fig. 1. Oviposition responses (meanSE) of P. protenor strates without drumming was not included. The demetrius to the foliage of five rutaceous plants. Cd: Citrus oviposition response to each sample was finally depressa, Ta: Toddalia asiatica, Em: Evodia meliifolia, Mt: represented as the mean percentage of responses Melicope triphylla, Mp: Murraya paniculata. recorded from more than 10 females. For test samples of a binary mixture, the significance of the dif- (33.8% and 33.7%, respectively), appeared to be ference in response between Cd-3 and the test sam- marginally acceptable to ovipositing females. ple was analyzed by a t-test (Aspin-Welch method). Cd-3 was chosen as the base component, because Oviposition response to extracts and fractions it displayed potent oviposition-stimulatory activity Female responses to methanol extracts and their (89.4%) and its chemical profile (Murakami et al., fractions prepared from individual plants were unpublished) closely resembled that of C. natsu- tested at three doses (0.5%, 1.0%, and 2.0%), ex- daidai. cept for C. depressa and T. asiatica, which evoked Larval feeding tests. Neonates and fifth-instar significant positive responses from females at as larvae, provided with young leaves of any one of low as 0.5% (Fig. 2). Oviposition responses to the the five rutaceous plants, were reared at 25°C extracts were nearly consistent with those to fo- under a photoregime of 16L : 8D to examine the liage, thereby indicating that methanol extracts rate of survival to the next stadium, which would contain key substance(s) responsible for host ac- serve as an estimate of larval fitness on these plants ceptance or rejection by females. It is apparent that (Courtney and Kibota, 1990). Fifth-instar larvae the two plants, C. depressa and T. asiatica, possess subjected to feeding experiments had been raised significant oviposition stimulant(s) and that the on potted Citrus plants (mainly C. natsudaidai) major chemical substance(s) inducing oviposition until fourth-larval ecdysis. Thirty neonates and ten are localized in aqueous fractions (Cd-3 and Ta-3). fifth-instar larvae were tested for each plant. The In addition, Cd-2, Ta-1, and Ta-2 also exerted a significance of the difference in larval survival was moderate to weak stimulatory effect on egg-laying, analyzed by a c 2-test. suggesting the co-occurrence of other stimulants in these plants. Females moderately responded to the extract of E. meliifolia and Em-3 at 0.5% (59.1% RESULTS and 65.3%, respectively), which implies that Em-3 Oviposition response to foliage contains oviposition stimulant(s) with considerable The acceptability of the five plants for oviposit- activity, although the foliage only weakly stimu- ing females of P. protenor demetrius was first lated egg-laying (Fig. 1). The oviposition response tested to assess their potential use as host plants to the extract, however, tended to dwindle as the (Fig. 1). Females exhibited potent positive re- dose of the sample increased. On the other hand, sponses to the foliage of C. depressa and also to T. females made little or almost no response to the asiatica. In marked contrast, Mu. paniculata was extract and each fraction of Me. triphylla. Since almost completely rejected, while E. meliifolia and these fractions failed to stimulate egg-laying even Me. triphylla, to which females responded weakly at higher concentrations, the unresponsiveness of 124 M. CHACHIN et al.

Fig. 2. Oviposition responses (meanSE) of P. protenor demetrius to methanolic extracts and fractions prepared from ﬁve rutaceous plants. females may be ascribed to either the absence of Oviposition response to binary mixtures stimulant(s) or the presence of deterrent(s). Re- The next experiments were designed to examine garding Mu. paniculata, weak positive responses whether oviposition deterrents are present in the (21–41%) were evoked by Mp-3 at all concentra- three plants, E. meliifolia, Me. triphylla, and Mu. tions, and moderate responses (51.5–56%) by the paniculata that were barely accepted or rejected by extract at concentrations higher than 1.0%. This in- females. Oviposition responses to their methanol dicates that the plant, although showing very poor extracts and fractions were thus tested in combina- acceptability, probably contains weak stimulant(s), tion with an equivalent amount of Cd-3 that dis- at least in Mp-3. played potent stimulatory activity (89.4%), so that Butterﬂy Host Selection 125

Fig. 4. Survival of first- and fifth-instar larvae on five rutaceous plant species. Fifth-instar larvae were transferred from Citrus plants (mainly C. natsudaidai) immediately after fourth-larval ecdysis to the respective plants. The results on C. depressa and E. meliifolia were not significantly different from each other (c 2-test).

deterrent effect on oviposition; rather, they seemed to be slightly stimulative, although only the extract of E. meliifolia and Em-1 weakly but signiﬁcantly deterred oviposition (p0.05). Although we have no clear explanation at present for the curious phe- nomenon shown in Fig. 2 that the oviposition response to E. meliifolia extract was inversely pro- portional to the sample concentration, this may be accounted for in part by the idea that the negative activity evoked by deterrent(s) (probably present mainly in Em-1) outweighed the positive activity evoked by stimulant(s) (probably present in Em-3) at higher concentrations (1.0% and 2.0%) probably due to the difference in dose dependency of activities between deterrent(s) and stimulant(s). Alterna- tively, the content of deterrent(s) in the extract (0.5%) might have been below the threshold level of detection by females.

Larval survival on five rutaceous plants The suitability of five plants for larval growth was appraised by examining the survival of first- Fig. 3. Oviposition responses (meanSE) of P. protenor and fifth-instar larvae fed with these plants (Fig. 4). demetrius to samples of binary mixture. Each sample was ad- Larvae, regardless of age, performed very well on mixed with an equivalent quantity of Cd-3 (0.5% each). The C. depressa. Unexpectedly, both first- and fifth-in- difference in response between Cd-3 (0.5%) and the test sam- star larvae, however, never survived on T. asiatica, ple was significant at p 0.05 (*) (t-test, Aspin-Welch which ovipositing females accepted as readily as C. method). depressa. Similarly, larvae of both instars failed to grow on Me. triphylla and Mu. paniculata, which is we may estimate to what extent they are responsi- compatible with the apparent avoidance of these ble for host recognition or preference by females. plants by females. On the other hand, the survival As shown in Fig. 3, most extracts and fractions de- of both instars fed with E. meliifolia, which fe- rived from the three plants were found to exert no males barely accepted, were as high as those fed 126 M. CHACHIN et al. with C. depressa, with neither feeding deterrence of each plant (Fig. 2). This finding is consistent nor growth-inhibitory effect. The high mortality of with earlier work in that most oviposition stimu- larvae on T. asiatica and Mu. paniculata seemed to lants for papilionid butterflies so far identified are be mainly due to the inhibition of feeding caused water-soluble compounds (Honda and Nishida, by some as yet uncharacterized anti-feedant(s) 1999; Ono et al., 2000a, b; Nakayama et al., 2003; present in the plants, since diet consumption by the Honda, 2005). In addition, it is intriguing that larvae was very low. In contrast, larvae fed with some non-aqueous fractions, Cd-2, Ta-1, and Ta-2, Me. triphylla vomited soon after gnawing a small were also stimulative to a limited extent, which fur- amount of its leaves. Therefore, the plant seems to ther suggests a synergistic system in the elicitation contain some toxic substances. of oviposition analogous to other Papilio butterflies (Honda, 1990; Ohsugi et al., 1991). Marginal acceptance of E. meliifolia by ovipositing females DISCUSSION seems to be ascribed to the coexistence of both Among the five plants tested, all but E. meliifolia moderate stimulant(s) and weak deterrent(s) (Figs. are distributed in allopatry with the habitat of P. 2 and 3), of which positive and negative stimuli protenor demetrius. The present result that the but- might have concurrently worked to eventually re- terfly is not fully adapted to most plants tested, ap- lease a weak positive response. Mu. paniculata, al- pears to reflect this situation. Apparently, C. de- though almost rejected by females, proved to con- pressa is most likely suitable for both oviposition tain weak stimulant(s) in the aqueous fraction (Mp- and larval growth. This is probably because the 3, Fig. 2) and no appreciable deterrent(s) in any phytochemical profile of the plant is very similar to fractions (Fig. 3). Moreover, its methanol extract that of C. natsudaidai (Honda, 1986; Murakami et evoked a moderate oviposition response at higher al., unpublished), which is one of the major host concentrations. This discrepancy is strongly sug- plants of the butterfly. Interestingly enough, gestive of the involvement of some volatile com- whereas ovipositing females readily accepted T. pound(s), probably lost during the process of sol- asiatica, no larvae were able to grow on this plant, vent evaporation, that would otherwise have ex- indicating that females are pre-adapted to the plant, erted a significant oviposition-deterring activity. but larvae cannot overcome its chemical barrier. The poor acceptability of Me. triphylla seems Although P. protenor demetrius and E. meliifolia to be explained by the fact that the plant contains are distributed sympatrically in some temperate re- neither appreciable oviposition stimulant(s) nor de- gions (Shikoku and Kyushu, for example) of Japan, terrent(s). The stimulatory or deterrent activities in females marginally accepted the plant for oviposi- egg-laying of individual fractions from five plants tion. Therefore, utilization of E. meliifolia as a host are summarized in Table 1. by the butterfly seems unlikely or would be far less Our previous study of the affinity of P. protenor frequent, if at all, in an environment where more demetrius for four other rutaceous plants, consist- preferred plants co-occur. It is, however, evident ing of C. unshiu, F. ailanthoides, P. amurense, and that not all but the majority of larvae deposited on O. japonica (Honda and Hayashi, 1995a), demon- the plant by chance or by females may, nonethe- less, grow into pupae. Consequently, whereas lar- Table1. Stimulatory or deterrent effect of extracts vae seem to be fully pre-adapted to E. meliifolia, and fractions derived from five rutaceous plants ovipositing females are insufficiently adapted to on oviposition by Papilio protenor demetrius the plant. This result contrasts strikingly with that Plant species Extract Fr. 1 Fr. 2 Fr. 3 of T. asiatica. On the other hand, the present findings clearly show that P. protenor demetrius is en- Citrus depressa S(***) S(*) S(***) tirely maladapted to both Me. triphylla and Mu. Toddalia asiatica S(***) S(*) S(*) S(***) paniculata. Evodia meliifolia S(**), D(*) D(*) S(**) In both C. depressa and T. asiatica on which fe- Melicope triphylla Murraya paniculata S(*) S(*) males readily oviposited (Fig. 1), dominant active substance(s) crucial for host recognition are S, stimulatory activity; D, deterrent activity. deemed to be present in the aqueous fraction (Fr. 3) ***, strong; **, moderate; *, weak. Butterfly Host Selection 127 strated that, of these, the most suitable plant was C. distributed from Honshu to the Amami Islands, is unshiu and the least suitable, O. japonica. The not a subspecies established by northward invasion present and previous findings, taken altogether, of a population, designated as P. protenor liukiuen- suggest that the nine rutaceous plants tested are sis, inhabiting Okinawa and Yaeyama Islands. In roughly arrayed as follows in decreasing order of fact, a pilot experiment with larvae of P. protenor acceptability for ovipositing females: C. unshiu liukiuensis derived from a female captured on Iri- C. depressaF. ailanthoidesT. asiaticaE. omote Island showed that considerable proportions meliifoliaMe. triphyllaMu. paniculataP. amu- of both neonates and fifth-instar larvae successfully renseO. japonica. The last two plants, in particu- grew into the next stadia on T. asiatica. lar, are characterized as having oviposition deter- Although P. protenor demetrius larvae are also rent(s) with moderate to potent intensity. For exam- found, on rare occasions, feeding on a few Zan- ple, phellamurin (a prenylated flavonoid glucoside) thoxylum species in the field, these are not its main present in P. amurense strongly deters egg-laying host plants in temperate regions. However, to the by P. protenor demetrius (Honda and Hayashi, best of our knowledge, the host plant most often 1995b). For Papilio xuthus, which is also a sym- utilized by P. protenor liukiuensis in the Yaeyama patric Rutaceae feeder having a host range similar Islands is Zanthoxylum nitidum, a subtropical/trop- to that of P. protenor demetrius, a flavonol glyco- ical shrub usually growing in a forest. Further side and two hydroxybenzoic acid derivatives pres- comparative investigations of the affinity of an- ent in O. japonica have been reported to exert sig- other subspecies, P. protenor liukiuensis, for the nificant deterrence against female oviposition above plants in combination with parallel phyto- and/or larval feeding (Nishida et al., 1990; Ono et chemical approaches to active substances regulat- al., 2004). On the other hand, in regard to larval ing host selection, would lead to a better under- growth, these plants may be arrayed as follows in standing of the physiochemical and sensory mech- decreasing order of suitability: C. unshiuC. de- anisms underlying the host range determination pressaF. ailanthoidesE. meliifoliaP. amurense and speciation of these two subspecies. O. japonicaT. asiaticaMu. paniculataMe. ACKNOWLEDGEMENTS triphylla. Recently, we reported the affinity of another This work was supported in part by a Grant-in-Aid for Sci- Rutaceae-feeding papilionid butterfly, Papilio poly- entific Research from the Japan Society for the Promotion of tes, for eight rutaceous plants including the five Science to K. Honda (No. 14560039). plants tested here and identified a few active com- REFERENCES pounds involved in the differential acceptance of Courtney, S. P. and T. T. Kibota (1990) Mother doesn’t know some of these plants (Nakayama et al., 2002, 2003; best: selection of hosts by ovipositing insects. In Insect- Murakami et al., 2003; Nakayama and Honda, plant Interactions. Vol. II (E. A. Bernays ed.). CRC 2004). An overall profile of oviposition responses Press, Florida, pp. 161–188. of P. polytes females to the five plants in question Feeny, P., E. Städler, I. Åhman and M. 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