Z. Naturforsch. 2015; 70(5-6)c: 145–150
Yong Zhang, Zhi-Hui Zhan, Shin-ichi Tebayashi, Chul-Sa Kim and Jing Li*
Feeding stimulants for larvae of Graphium sarpedon nipponum (Lepidoptera: Papilionidae) from Cinnamomum camphora DOI 10.1515/znc-2014-4155 Received September 2, 2014; revised January 20, 2015; accepted June 17, 2015
Abstract: The feeding response of larvae of the swallowtail butterfly, Graphium sarpedon nipponum (Lepidoptera: Papilionidae), is elicited by a methanolic extract from camphor tree (Cinnamomum camphora) leaves. Based on bioassay-guided fractionation, three compounds, isolated from the methanolic extract of fresh leaves of the camphor tree, were revealed to be involved in a multi-component system of feeding stimulants. Structures of these feeding stimulants were identified as sucrose, 5-O-caffeoylquinic acid and quercetin 3-O-β-glucopyranoside by NMR and LC-MS. Keywords: Cinnamomum camphora; feeding stimulants; Graphium sarpedon nipponum.
1 Introduction Most butterfly species are phytophagous and usually utilize a limited range of host plants in nature. Although the host range of an insect is determined by a diversity of ecological, geographical, physiological and behavioral factors, the key elements underlying the host range determination are phytochemicals [1, 2]. As for larval insects, plant chemicals released from the leaf typically influence the decision as to whether or not the feeding will be continued [3, 4]. A number of swallowtail butterfly species in the genus Graphium commonly feed on species of the plant family *Corresponding author: Jing Li, Ninth People’s Hospital of Chongqing, Beibei District, Chongqing 400715, China, Fax: +86-023-6825-0994, E-mail: [email protected] Yong Zhang: College of Resources and Environment, Southwest University, Beibei District, Chongqing, China Zhi-Hui Zhan, Shin-ichi Tebayashi and Chul-Sa Kim: Faculty of Agriculture, Department of Agriculture, Kochi University, B200 Monobe, Nankoku, Japan
Lauraceae, including the cinnamon tree. This insect belongs to the tribe Graphiini, which is placed between the Troidini and Papilionini in the evolutionary tree [5]. Thus, chemicals related to the host selection of species of the genus Graphium should be given attention to elucidate evolutionary processes in the Papilionidae family. Graphium sarpedon nipponum, the common bluebottle, is one of the Lauraceae-feeding species, which usually utilizes Cinnamomum camphora as its major host. Adults of this butterfly are attracted by volatile components from C. camphora, which have been found to act as olfactory cues in determining the choice of food plants by G. sarpedon nipponum [6]. For a better understanding of the physiochemical background of present-day host utilization and host range evolution in Graphium butterflies, further attempts are needed to investigate the chemical basis for differential acceptance of a potential host plant. Previously we reported the isolation and characterization of α-linolenic acid as a feeding stimulant from the hexane fraction of a C. camphora methanolic extract [5]. As part of our ongoing study on feeding stimulants from C. camphora, we have now investigated the polar fraction of C. camphora fresh leaves and isolated three additional feeding stimulants for G. sarpedon nipponum.
2 Materials and methods 2.1 Insect and plant Eggs of G. sarpedon nipponum were collected from young branches of C. camphora trees growing on Monobe Campus of Kochi University, Japan. Larvae were kept at 25±3 °C and 70% relative humidity in Petri dishes with a 15:9 h, L:D photoperiod. The animate fifth instar larvae were used for bioassay. Fresh leaves of C. camphora were collected from the same source and used for extraction and bioassay.
2.2 Bioassay The behavioral bioassay for the isolation of attractants and stimulants in the initiation of the feeding response were carried out as
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146 Zhang et al.: Feeding Stimulants from Cinnamomum camphora previously described [5]. Activities were expressed as gram leaf equivalents (g.l.e.) per semicircular Styrofoam test discs (45 mm in diameter, 0.7 mm in thickness). Samples of 0.5 g.l.e. were applied onto the discs. As the control, the same volume of a solvent blank was added to a disc. After drying, discs were placed in a Kimwipes box (13 cm × 12 cm × 9 cm) with the upper end open, and each disc was inserted into a slit of foam bottom, thereby kept in upright position. Usually one disc corresponded to one test sample, and the appropriate number of discs were introduced into the box for bioassay. Three fifth instar larvae, which had been starved for 6 h, were left in the box and their feeding behavior was observed for 24 h (25±3 °C, 15:9 h, D:L photoperiod). Styrofoam discs containing attractants or stimulants would be consumed by the larvae, and the feeding damage (consumed area in mm2) of discs was measured by a flat-bed scanner. Each test was repeated at least five times, and consumed areas of Styrofoam discs were averaged to serve as the index of the feeding stimulatory effect. Statistical analysis used one-way analysis of variance (ANOVA) followed by Tukey HSD test (p
Yong Zhang, Zhi-Hui Zhan, Shin-ichi Tebayashi, Chul-Sa Kim and Jing Li*
Feeding stimulants for larvae of Graphium sarpedon nipponum (Lepidoptera: Papilionidae) from Cinnamomum camphora DOI 10.1515/znc-2014-4155 Received September 2, 2014; revised January 20, 2015; accepted June 17, 2015
Abstract: The feeding response of larvae of the swallowtail butterfly, Graphium sarpedon nipponum (Lepidoptera: Papilionidae), is elicited by a methanolic extract from camphor tree (Cinnamomum camphora) leaves. Based on bioassay-guided fractionation, three compounds, isolated from the methanolic extract of fresh leaves of the camphor tree, were revealed to be involved in a multi-component system of feeding stimulants. Structures of these feeding stimulants were identified as sucrose, 5-O-caffeoylquinic acid and quercetin 3-O-β-glucopyranoside by NMR and LC-MS. Keywords: Cinnamomum camphora; feeding stimulants; Graphium sarpedon nipponum.
1 Introduction Most butterfly species are phytophagous and usually utilize a limited range of host plants in nature. Although the host range of an insect is determined by a diversity of ecological, geographical, physiological and behavioral factors, the key elements underlying the host range determination are phytochemicals [1, 2]. As for larval insects, plant chemicals released from the leaf typically influence the decision as to whether or not the feeding will be continued [3, 4]. A number of swallowtail butterfly species in the genus Graphium commonly feed on species of the plant family *Corresponding author: Jing Li, Ninth People’s Hospital of Chongqing, Beibei District, Chongqing 400715, China, Fax: +86-023-6825-0994, E-mail: [email protected] Yong Zhang: College of Resources and Environment, Southwest University, Beibei District, Chongqing, China Zhi-Hui Zhan, Shin-ichi Tebayashi and Chul-Sa Kim: Faculty of Agriculture, Department of Agriculture, Kochi University, B200 Monobe, Nankoku, Japan
Lauraceae, including the cinnamon tree. This insect belongs to the tribe Graphiini, which is placed between the Troidini and Papilionini in the evolutionary tree [5]. Thus, chemicals related to the host selection of species of the genus Graphium should be given attention to elucidate evolutionary processes in the Papilionidae family. Graphium sarpedon nipponum, the common bluebottle, is one of the Lauraceae-feeding species, which usually utilizes Cinnamomum camphora as its major host. Adults of this butterfly are attracted by volatile components from C. camphora, which have been found to act as olfactory cues in determining the choice of food plants by G. sarpedon nipponum [6]. For a better understanding of the physiochemical background of present-day host utilization and host range evolution in Graphium butterflies, further attempts are needed to investigate the chemical basis for differential acceptance of a potential host plant. Previously we reported the isolation and characterization of α-linolenic acid as a feeding stimulant from the hexane fraction of a C. camphora methanolic extract [5]. As part of our ongoing study on feeding stimulants from C. camphora, we have now investigated the polar fraction of C. camphora fresh leaves and isolated three additional feeding stimulants for G. sarpedon nipponum.
2 Materials and methods 2.1 Insect and plant Eggs of G. sarpedon nipponum were collected from young branches of C. camphora trees growing on Monobe Campus of Kochi University, Japan. Larvae were kept at 25±3 °C and 70% relative humidity in Petri dishes with a 15:9 h, L:D photoperiod. The animate fifth instar larvae were used for bioassay. Fresh leaves of C. camphora were collected from the same source and used for extraction and bioassay.
2.2 Bioassay The behavioral bioassay for the isolation of attractants and stimulants in the initiation of the feeding response were carried out as
Brought to you by | Colorado State University Authenticated Download Date | 12/10/15 2:36 AM
146 Zhang et al.: Feeding Stimulants from Cinnamomum camphora previously described [5]. Activities were expressed as gram leaf equivalents (g.l.e.) per semicircular Styrofoam test discs (45 mm in diameter, 0.7 mm in thickness). Samples of 0.5 g.l.e. were applied onto the discs. As the control, the same volume of a solvent blank was added to a disc. After drying, discs were placed in a Kimwipes box (13 cm × 12 cm × 9 cm) with the upper end open, and each disc was inserted into a slit of foam bottom, thereby kept in upright position. Usually one disc corresponded to one test sample, and the appropriate number of discs were introduced into the box for bioassay. Three fifth instar larvae, which had been starved for 6 h, were left in the box and their feeding behavior was observed for 24 h (25±3 °C, 15:9 h, D:L photoperiod). Styrofoam discs containing attractants or stimulants would be consumed by the larvae, and the feeding damage (consumed area in mm2) of discs was measured by a flat-bed scanner. Each test was repeated at least five times, and consumed areas of Styrofoam discs were averaged to serve as the index of the feeding stimulatory effect. Statistical analysis used one-way analysis of variance (ANOVA) followed by Tukey HSD test (p
