100118 (347)

Biosci. Biotechnol. Biochem., 74 (10), 100118-1–4, 2010

Volatile Attractants for the Common Bluebottle, sarpedon nipponum, from the Host,

y Jing LI, Ryu WAKUI, Shin-ichi TEBAYASHI, and Chul-Sa KIM

Department of Agriculture, Faculty of Agriculture, Kochi University, B200 Monobe, Nankoku 783-8502,

Received February 17, 2010; Accepted June 21, 2010; Online Publication, October 7, 2010 [doi:10.1271/bbb.100118]

Floral scent has been shown to elicit behavioral The common bluebottle, Graphium sarpedon (Lep- responses by butterflies which forage for flowers after idoptera: Papilionidae), is a species of swallowtail receiving appropriate signals. In comparison with inves- butterfly distributed throughout South and Southeast tigations about the role of floral scent, those of foliar odor Asia. There are approximately 15 subspecies with are, however, very few. In this study, the foliar volatiles of differing geographical distributions. One of the subspe- Cinnamomum camphora (), which had been cies, G. sarpedon nipponum, inhabits mainly southern collected by air entrainment, exhibited activities toward Japan and the adult butterfly appears from May to Graphium sarpedon nipponum (Papilionidae) in both October each year. This butterfly is a Lauraceae-feeding electrophysiological and behavioral tests. The volatiles butterfly, especially preferring Cinnamomum camphora. were analyzed by capillary gas chromatography with Many males and females are commonly observed electro-antennographic detection (GC-EAD). Two electro- around this tree during mating, ovipositing and feeding physiologicalAdvanceactive compounds were found which View were on the nectar. determined as nonanal and decanal by using gas chroma- These observations led us to study the attractant(s) to tography-mass spectrometry (GC-MS). Female butterflies G. sarpedon nipponum from the foliage of C. camphora. generally tend to show a greater EAG response than males The role of the volatiles from the foliage of C. camphora to the headspace volatiles and EAG-active aldehydes. Two was studied by electro-antennography (EAG) and gas EAG-active aldehydes were found in attractant tests to chromatography coupled with electro-antennographic be attractive to both sexes of the butterfly when treated detection (GC-EAD). The screened EAD-active com- individually. Although the difference between the sexes pounds were further used to check their behavioral was not significant, the female butterflies’ preference activity in an outdoor attractant bioassay. tended to be more active than that of the males. Materials andProofs Methods Key words: attractant; Graphium sarpedon nipponum; Cinnamomum camphora; nonanal; decanal Plants and . Adults of the common bluebottle, G. sarpedon nipponum, approaching and hovering on camphor trees, were collected The sequence of behavior for most phytophagous from July to October 2008 on the Monobe campus of Kochi University, Japan. The butterflies for the attractant test were used insects during host plant selection is often in three immediately after catching, and the other butterflies for the electro- 1) different stages: orientation, approach, and assessment. physiological test were maintained for a day in paper bags at 25 C In trying to locate the habit of its host plant during without being fed. Foliages samples of C. camphora were also orientation, olfactory stimuli alone, or in combination collected on the Monobe campus of Kochi University. The cut end with visual cues are used by insects. These stimuli of a branch was dipped in water during the procedure of air prompt the to initiate upwind flight. In the second entrainment and used for the attractant bioassay. step, the approach to the host plant may be triggered by an olfactory cue or by olfactory and visual cues. Headspace volatile collection. Freshly cut branches, each set in a 50-ml flask with water, were placed in a 20-liters glass desiccator for Olfactory cues play an important role for many insects one week. The branches were changed to fresh ones every day. Air, 2) in the first two behavioral steps. after being purified by passing through an Orbo100 tube (350/ Butterflies of the family Papilionidae are among the 175 mg carbotrap, 7 mm i.d., 110 mm length; Supelco, USA), was most common insect taxa.3) Research on this family has introduced into the desiccator and the volatiles were trapped in another focused mainly on secondary metabolites,4–8) which Orbo100 tube. The trapped volatiles were eluted from the Orbo100 often act as oviposition and feeding stimulants.9–11) tube with 250 ml of distilled diethyl ether. The yielded extract was These metabolites include amino acid derivatives, sealed in glass vials and stored at 20 C in a freezer prior to being analyzed. sugar-related acids, alkaloids, flavonoids, and hydroxy- 12) cinnamic acid derivatives. However, there have been Electro-antennograms (EAGs). After being briefly anesthetized few chemical ecological studies on the volatile com- with ice, one antenna of the male or female butterfly was excised with pounds13) relating to orientation and approach. To the microsurgical scissors, and a 1/3 segment was cut off from the tip of best of our knowledge, investigations on volatile com- the antenna. The cut antenna was then fixed between two stainless pounds has been largely reported for odors from the electrodes by pushing the base and tip into droplets of electrical floral scent,14–22) studies on the volatiles from foliage as conductive gel (Spectra 360 electrode gel: Parker, Orange, NJ, USA). 23,24) A 20-ml amount of each test sample (nonanal, and decanal at different an attractant being rare. concentrations and the headspace volatiles) in diethyl ether was

y To whom correspondence should be addressed. Fax: +81-88-864-5186; E-mail: [email protected] 100118-2 J. LI et al. applied to a filter paper strip (10 mm 4mm). After removing the solvent, the filter paper was inserted into a Pasteur pipette and a test sample on the filter paper was introduced on to the antenna by a short 15.65 min puff using a CS-01 stimulus air controller (0.1 s duration; Syntech, 19.89 min Hilversum, The Netherlands). All the EAG responses were recorded in FID signal a computer through an IDAC 02 interface (Syntech) with the Electroantennography version 2.5 software program (Syntech). Three antennae from 3 males and 3 antennae from 3 females were each used for the headspace volatiles in the EAG test. The yielded EAG values were averaged. In the dose-response trial, 5 antennae from EAD signal 5 females and 4 antennae from 4 males were each used for the EAG activity of nonanal, while 4 antennae from 4 females and 3 antenae from 3 males were each used for decanal. EAD signal Gas chromatography with electro-antennographic detection (GC- EAD). The electro-antennographic activity of the volatile extract of C. camphora was analyzed with a gas chromatograph equipped with an electro-antennographic detector using 5 antennae from 5 females and 4 10 15 20 25 antennae from 4 males. A Hewlett-Packard 6890 GC (Hewlett- Retention time (min) Packard, Wilmingtom, DE, USA) was equipped with an HP-5 column (30 m 0:33 mm i.d. 0.25 mm film thickness, crosslinked 5% PH ME Fig. 1. Simultaneous EAD and FID Responses of Both Male and Siloxane). The column temperature was programmed from 40 C for Female G. sarpedon nipponum to the Volatile Extract of C. camphora. 1 min, and then 5 C/min to 250 C. The eluent from the column was split into two lines, one of each leading to a flame ionization detector (FID) and EAD at a ratio of 1:1. The outlet for the EAD was held in a 100 mV from both sexes. The average EAG response of humidified air stream flowing at 300 ml/min over an antennal the female butterfly (411 mV) tended to be stronger than preparation which had been prepared by the method already described. that of the male (360 mV), although the difference was The compounds eluted from the capillary column were delivered to the Advance View p < : antenna through a glass tube. The antennal signals were stored and not significant ( 0 05). analyzed by a PC equipped with a serial IDAC 02 interface box and the The GC-EAD analysis of the headspace volatiles EAD version 2.5 program (Syntech, Hilversum, the Netherlands). compounds from C. camphora (Fig. 1) revealed that two compounds, A (Rt 15.65 min) and B (Rt 19.89 min), Gas chromatography-mass spectrometry. The antennal active peaks consistently elicited electrophysiological responses from from the volatile extract of C. camphora were identified by GC-MS the antennae of the male and female G. sarpedon [Hewlett-Packard 6890 GC coupled to a JMS-600 mass spectrometer nipponum. The mass spectrum of compound A (Rt (Jeol, Tokyo, Japan)] with an HP-5 column (30 m 0:33 mm i.d., þ 0.25 mm film thickness, crosslinked 5% PH ME Siloxane). The oven 15.65 min) showed a base peak at m=z 57, M at m=z þ program and carrier gas were the same as those for the GC-EAD 142, diagnostic fragment ions at m=z 124 [M H2O] analysis. Ionization was achieved in the electro-impact mode at 70 eV and 113 [M CHO]þ, and a McLafferty rearrangement þ and 250 C. The compounds were identified by comparing their mass peak at m=z 98 [MProofs C2H4O] , suggesting the presence spectra and retention times with those of authentic samples. Two of a double bond and -H, all of which indicated a C9 authentic standards, nonanal and decanal, were purchased from TCI aldehyde. Compound B (Rt 19.89 min), with a base peak (Tokyo, Japan). Their respective purity was above 99% and 99.5%. at m=z 57, Mþ at m=z 156, diagnostic fragment ions at Attractive bioassay. The behavioral bioassays were conducted in a m=z 138 and 127, and a McLafferty rearrangement peak þ net cage (5m 5m 3m in height) from August to October 2008 at at m=z 112 [M C2H4O] , was assumed to be a C10 the Monobe campus of Kochi University, Japan. Ten ml of nonanal and aldehyde. The mass spectra and GC retention times of 10 ml of decanal were separately applied to filter papers (20 mm compounds A and B coincided well with those of 7mm). The two treated filter papers and one untreated filter paper nonanal and decanal, respectively. Accordingly, com- (control) were respectively put on the stems of three twigs kept in pound A and compound B were respectively identified 20-ml vials with water. The prepared three lures were hung in the cage at a 2-m height from the ground and at 1-m intervals. A butterfly was as nonanal and decanal. introduced into the cage and its flight behavior was observed for The sensitivity of G. sarpedon nipponum to different 30 min. The flight was recorded as a positive response when the concentrations of the two compounds was analyzed by butterfly touched a twig. Trials were replicated three times for each using EAG. A total of 16 antennae was used. As shown butterfly, changing the arrangement of the twigs. The responses of in Fig. 2, the responses of both male and female individuals to the given samples were averaged. A total of 7 males and antennae to nonanal and decanal were dose-dependent. 8 females was used. No significant difference in the EAG responses was apparent between the sexes in all ranges of these two Statistical analyses. Student’s t-test (p < 0:05) was used to analyze the electrophysiological activity of the extracts. A one-way analysis aldehydes. Although the females tended to be more of variance (ANOVA) followed by the t-test were conducted for the sensitive to nonanal for most dosages, the difference other tests. between nonanal and decanal was not significant in both sexes (Fig. 2). Results The attractive bioassay was conducted by using 7 male and 8 female butterflies towards the two aldehyde- A total of 322 g of fresh foliage (46 g of foliage per treated twigs and one untreated twig as a control. After a day) of C. camphora was used for volatile collection. butterfly had been introduced into the cage, the flight The antennal response of G. sarpedon nipponum to 26 g behavior of the butterfly was strongly affected by the of foliage equivalent per filter paper (g.f.e./fp)] in the presence of the twigs. The average times of touching by EAG test was significantly stronger than the air control females and males to the control twig were significantly (p < 0:05) which elicited a small EAG response below lower than those to the twigs treated by an aldehyde for Attractant for Graphium sarpedon nipponum from Cinnamomum camphora 100118-3

350 18 Male (n = 7) Nonanal 300 16 Female (n = 8) 14 250 (n = 5) 12 V) µ 200 (n = 4) 10

150 8

6

EAG response ( EAG 100

Number of touch times 4 50 2

0 0 -4 -3 -2 -1 0 1 2 3 10 10 10 10 10 10 10 10 Control Decanal Nonanal Dose (µg) Treatment

350 Fig. 3. Number of Touch Times by G. sarpedon nipponum to Twigs Decanal Treated with the EAD-Active Compounds. 300

250 and female butterflies in either the electrophysiological

V) or behavioral test, support males searching for females µ 200 around the host plant, and they can therefore be expected (n = 3) to have evolved similar sensitivity to the females to 150 (n = 4) host-plant chemicals. The result of that the female Advance response ( EAG 100 Viewbutterflies’ preference tended to be more active than that of the males in both the electrophysiological and 50 behavioral tests indicates the greater olfactory need of the female butterflies. 0 10-4 10-3 10-2 10-1 100 101 102 103 Two semiochemicals, nonanal and decanal, which Dose (µg) were identified by GC and GC-MS, are well known minor components of the green leaf order. Previous Fig. 2. Electro-Antennogram (EAG) Responses of the Female ( ) reports have indicated these aldehydes as EAG active and Male ( ) Antennae of G. sarpedon nipponum to Various Doses compounds toward Lepidopterous stemborers, Chilo of Nonanal and Decanal. partellus and Busseola fusca, from their host plants, Zea mays, Sorgum bicolor, Pennisetum purpureum and Hyparrheria tambaProofs.29) More than 30 EAG active com- Table 1. Number of Touch Times by the Female and Male pounds, including nonanal and decanal, from Aesculus Butterflies hippocastanum leaves fed on by larva of Cameraria Female (n ¼ 8) Male (n ¼ 7) ohridella have previously been reported. These two aldehydes were revealed to be oviposition inhibitors Control 4:8 1:10:9 0:3 against adult C. ohridell.30,31) Nonanal 12:4 2:18:0 1:2 Decanal 10:5 2:15:0 1:2 The number of touch times for the male and female in the attractive bioassay, increased in the order of (Average SE) control < decanal < nonanal. It is considered that both the male and female use nonanal and decanal as both sexes (p < 0:01). The attractive strength of the attractants for finding the host location. However, the twigs treated with nonanal towards G. sarpedon nippo- total number of touch times by the female butterfly num tended to be slightly stronger than that of the twigs tended to be higher than that of the male, indicating that treated by decanal for both sexes as shown in the the female butterfly had greater adaptation than the Table 1. There was no significant difference between male. The results may reflect the greater need of the sexes, although the females generally tended to show females to be highly sensitive to chemicals in their more activity than the males (Fig. 3). surroundings because of their biological obligation to ensure the survival of their offspring.32) Discussion The optimum concentrations of both compounds in combination must be investigated in the future since Early instar larvae of Papilio species are generally they clearly have attraction activity toward G. sarpedon unable to move from plant to plant, so host-plant selec- nipponum. tion is determined mainly by the mother females.25,26) The female is sensitive to the host plant because of her Acknowledgment involvement in host location for oviposition. The male is also frequently found around the host in search of This study was partially supported by gran^aid for females.27,28) This behaviour suggests that males may young scientists (B), 21780048, in 2009 from the also have evolved the ability to respond to host-plant Ministry of Education, Culture, Sports, Science, and volatiles. Our results, which show that there were no Technology. significant sexual differences between male butterflies 100118-4 J. LI et al. References 17) Honda K, Oˆ mura H, and Hayashi N, J. Chem. Ecol., 24, 2167– 2180 (1998). 1) Renwick J, Experientia, 45, 223–228 (1989). 18) Oˆ mura H, Honda K, and Hayashi N, J. Chem. Ecol., 31, 1895– 2) Hartlieb E and Anderson P, ‘‘Insect Olfaction,’’ ed. Hansson BS, 1906 (2005). Springer-Verlag Press, Heidelberg, Berlin, pp. 315–349 (1999). 19) Oˆ mura H, Honda K, and Hayashi N, J. Chem. Ecol., 26, 655– 3) Silva-Branda KL, Freitas AVL, Brower AVZ, and Solferini VN, 666 (2000). Mol. Phylogenet. 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