Chemical Factors Influencing the Feeding Preference of Three

Appl. Entomol. Zool. 40 (1): 161–168 (2005) http://odokon.ac.affrc.go.jp/

Chemical factors inﬂuencing the feeding preference of three Aulacophora leaf beetle species (Coleoptera: Chrysomelidae)

Makoto ABE*,† and Kazuhiro MATSUDA Insect Science and Bioregulation Laboratory, Faculty of Agriculture, Tohoku University; Sendai, Miyagi 985–8555, Japan (Received 23 July 2004; Accepted 2 November 2004)

Abstract Three cucurbitaceous feeding leaf beetle species, Aulacophora indica, A. lewisii, and A. nigripennis, were investigated with regard to the relationship between feeding preference by the beetles and cucurbitacins B, E, I, and E-glucoside contents in the leaves of 18 cucurbitaceous plant species. HPLC analysis and TLC analysis of A. lewisii revealed that the cucurbitacin B and E-glucoside, which are strong feeding stimulants for A. indica and A. lewisii, were abundant in Lagenaria siceralia, Sicyos angulatus, and Cucumis melo leaves. In a feeding preference test using fresh leaves, A. indica preferred various cucurbitaceous plant species and methanol extracts regardless of the contents of the four cucurbitacins. A. lewisii mostly fed on Luffa cylindrica leaves in which the four cucurbitacins were not detected. A. lewisii was also stimulated to feed by the methanol extracts of Lagenaria siceraria and L. cylindrical, while A. nigripennis, which is only slightly stimulated to feed by the four cucurbitacins, fed primarily on Trichosanthes kirilowii var. japonica leaves. Among 18 cucurbitaceous plants, only Diplocyclos palmatus leaves were completely rejected by the three beetle species, and these beetle species did not even feed on D. palmatus leaves treated with the cucurbitacins. These results indicate that some leaf constituents as well as the four cucurbitacins play an important role in host plant selection by the three cucurbitaceous feeding beetle species.

Key words: Cucurbitaceae; cucurbitacin; Aulacophora; feeding stimulant; feeding deterrent

lewisii Baly (Abe et al., 2000). On the contrary, A. INTRODUCTION lewisii was strongly stimulated to feed by the cu- Cucurbitacins are known as feeding stimulants curbitacins, but the beetle feeds almost solely on for cucurbitaceous feeding leaf beetles such those leaves from the genus Luffa (Lewis and Metcalf, in the genera Acalymma, Aulacophora, Ceratoma, 1996; Abe et al., 2000). For these reasons, we have and Diabrotica, and are regarded as an essential come to believe that some other constituents in ad- factors for these beetle species to recognize the cu- dition to the cucurbitacins in leaves of cucurbita- curbitaceous host plants (Chambliss and Jones, ceous plants play important roles in the host plant 1966; Shinha and Krishna, 1969, 1970; Metcalf et selection by the Aulacophora beetles. Although cu- al., 1980; Ferguson et al., 1983; Nishida et al., curbitacins have been analyzed on cotyledons, 1986; Metcalf and Metcalf, 1992; Eben et al., fruits, and roots of many cucurbitaceous plants 1997). On the other hand, we have reported an ex- (Rehm et al., 1957; Rehm and Wessels, 1957; ample for which the host plant selection by two Lavie and Glotter, 1971; Metcalf et al., 1982; Fer- Aulacophora beetle species is explained by the guson et al., 1983; Nishida et al., 1986; Kitajima presence of cucurbitacins in the cucurbitaceous and Tanaka, 1989), investigations of the content of host plant leaves: A. nigripennis Motschulsky uses the cucurbitacins in leaves are limited. As for Aula- genus Trichosanthes plants (Cucurbitaceae) as cophora beetle species, the relationship between hosts, however, their feeding responses to cucur- host plant preference and the cucurbitacins is not bitacins B, E, I, and E-glucoside were much clear. Elucidation of host plant selection mecha- weaker than those of A. indica (Gmelin) and A. nisms will contribute to agricultural applications

*To whom correspondence should be addressed at: E-mail: [email protected] † Present address: Final Disposal Engineering Section, Research Center for Material Cycles and Waste Management, National Institute for Environ- mental Studies; Tsukuba, Ibaraki 305–8506, Japan DOI: 10.1303/aez.2005.161

161 162 M. ABE and K. MATSUDA such as the breeding of insect resistant plants as of Faculty of Agriculture, Tohoku University. well as increasing our understanding of insect- Chemicals. Cucurbitacins B, E, and E-gluco- plant relationships. To clarify the relationship be- side were isolated from the fruits of Ecuballium tween cucurbitaceous plants and Aulacophora bee- elaterium (L.) A. Rich. and identified by NMR and tles, we evaluated the four cucurbitacins in leaves MS spectrometer (Abe et al., 2000). Cucurbitacin I of cucurbitaceous plants and investigated leaf con- was purchased from Funakoshi Co. Ltd. stituents that play an important role in host plant Feeding preference for leaves. To evaluate the selection by three Aulacophora beetle species, A. feeding preference of the three beetle species for indica, A. lewisii, and A. nigripennis. cucurbitaceous plants, a feeding test was conducted with the 18 cucurbitaceous plants described above. A filter paper (7 cm diameter, No. 1, Toyo) was MATERIALS AND METHODS placed at the bottom of a petri dish (6 cm diameter) Insects. A. nigripennis was collected in a field at and distilled water was added to maintain humidity. the Faculty of Agriculture, Tohoku University. A. Two centimeter disks of fresh test plant leaves were indica and A. lewisii were collected at Ishigaki Is- punched out with a cork borer. Two such leaflet land, Okinawa Prefecture. Adults were kept in a pieces were weighed and then placed on the filter cylindrical plastic case (914 cm diameter) lined paper at the bottom of the petri dish. Three insects on the bottom with moistened paper towel, and lar- (mixed sex and starved for 24 h) were released into vae were in cylindrical plastic cases (59 cm di- the petri dish. The test was continued for 2 h and ameter) filled halfway with moistened sand. These replicated five times. After completion of the test, insects were successively reared at 241°C, the leaflets were removed and weighed again. The 16L–8D photoregime and provided cucurbitaceous amount fed on was defined as the weight con- plant leaves (A. indica: Cucurbita maxima Duch- sumed. esne, Sicyos angulatus L., and Trichosanthes kir- Feeding response to D. palmatus leaf treated ilowii Maxim var. japonica Kitam. leaves; A. with cucurbitacins. On D. palmatus, neither the lewisii: Luffa cylindrical (L.) Roemer leaf; A. ni- fresh leaf nor the methanol extract was preferred gripennis: T. kirilowii var. japonica and S. angula- by the beetles. Therefore, it was considered that D. tus leaves) as food. palmatus leaf either contains strong feeding deter- Plants. Eighteen cucurbitaceous plants (nine rents or lacks a feeding stimulant. To clarify the wild species, nine cultivated species) were used for presence of feeding deterrent or lack of feeding tests. The wild species were: Diplocyclos palmatus stimulant, a feeding test using D. palmatus leaf (L.) C. Jeffrey, Gynostemma pentaphyllum Makino, treated with cucurbitacins was conducted. As a Melothria japonica Maxim. ex Cogn., M. liukiuen- feeding stimulant, cucurbitacin B was used for A. sis Nakai, Schizopepon bryoniaefolius Maxim., S. indica, and cucurbitacin I for A. lewisii. These cu- angulatus, Trichosanthes bracteata (Lam.) Voigt, curbitacins are known to exhibit strong feeding T. kirilowii var. japonica, and T. rostrata Kitam. stimulant activity to the respective two species Cultivated species (cultivar name and the seed (Abe et al., 2000). Because A. nigripennis was only company) were as follows: Benincasa hispida minimally stimulated to feed by cucurbitacins, (Thunb.) Cogn. (Maokuwa, Fukutane Co. Ltd.), methanol extract of T. kirilowii var. japonica leaf Citrulus lanatus (Thunb.) Matsum. and Nakai was used as a feeding stimulant. A fresh 2 cm leaf (Shimabeni, Tohoku Co. Ltd.), Cucumis melo L. disk of D. palmatus was punched out. An ethanol (Ougon, Sakata Seed Co. Ltd.), Cucumis sativus L. solution (0.03 ml) of feeding stimulants was ap- (Suyo, Sakata Seed Co. Ltd.), Cucurbita maxima plied uniformly to the upper surface of the leaf (Miyako, Sakata Seed Co. Ltd.), Lagenaria sicer- disk. The solvent was removed quickly by air-dry- aria (Molina) Standl. (Sennari, Sakata Seed Co. ing. The concentration of the cucurbitacins was ad- Ltd.), Luffa cylindrical (L.) Roemer (Futonaga, justed to 0.1 mg/ml, and the concentration of Sakata Seed Co. Ltd.), Momordica charantia L. methanol extract of T. kirilowii var. japonica leaf (Futoreishi, Takii Seed Co. Ltd.), and Sechium was 1 g leaf equivalent/ml. One leaflet was edule (Jacq.) Sw. (Hayatouri, Fukutane Co. Ltd.). weighed and then placed on the filter paper at the All plants were cultivated at the experimental field bottom of the petri dish. Three insects (mixed sex Feeding Preference of Three Aulacophora Leaf Beetle Species 163 and starved for 24 h) were released into the petri ture of the water bath was less than 40°C). Chloro- dish. Each test was continued for 4 h and replicated form extract was then dissolved in chloroform ® five times. After completion of the test, the leaflet (0.2 ml) and placed on a Sep-pak C18 cartridge. was removed and weighed again. The amount fed After the solvent was removed by airflow, the ex- on was defined as the weight consumed. tract was eluated with 70% methanol in water Feeding preference to methanol extracts from (3 ml). The eluate was dried in vacuo and then dis- leaves. To confirm whether feeding stimulants were solved in methanol (0.2 ml) for test sample. The present in 18 cucurbitaceous plant leaves, feeding sample was subjected to HPLC and TLC analysis. responses by three beetle species to leaf extracts HPLC systems were JASCO GULLIVER SERIES were investigated. Fresh leaves were extracted with with DP-L 910 multi scan detector (Japan Spectro- methanol (20 ml/g fresh leaves, repeated three scopic Co. Ltd.) and TOSO CCPM pump with UV- times). The solvent was removed by a rotary evap- 8000 detector (TOSO Co. Ltd.). Wavelength was orator. Feeding assay was conducted with the filter adjusted to 200–400 nm (DP-L 910 multi scan de- paper procedure described by Abe et al. (2000). tector) and 254 nm (UV-8000 detector), respec- The test methanol solution was adjusted to 1g fresh tively. The analysis column was TSKgel® ODS-80 leaf equivalent/ml. Each test was replicated five Ts (TOSO, 1504.6 mm i.d.). A gradient separa- times. After completion of the test, each test filter tion was achieved using acetonitrile : water (2 : 8, paper was divided into 100 sections (each solvent A) and acetonitrile : water (6 : 4, solvent B) 22 mm) and the number of sections with bites in as eluents. The mixture ratio of solvent A : B was them was counted. The intensity of feeding was from 1 : 0 to 0 : 1 for 50 min, and the flow rate was represented as the feeding stimulant index (max 1 ml/min. Ten microliters of test solution was in- value: 200/dish). jected. Identification and quantification of cucur- Feeding response to host plant leaves treated bitacins were performed by comparison with stan- with D. palmatus extract. To clarify the presence dards of cucurbitacins B, E, E-glucoside, and I of feeding deterrent in the methanol extract from which are known constituents of many cucurbita- D. palmatus leaves, the feeding test using host ceous plants (Lavie and Glotter, 1971; Metcalf and plant leaf treated with the methanol extract from D. Metcalf, 1992). Recovery of cucurbitacins was also palmatus leaves was conducted at under no-choice measured by adding standard cucurbitacins of condition. A S. angulatus leaf disk (2 cm diameter) known concentrations to methanol extract of T. kir- was used for the test of A. indica, a L. cylindrical ilowii var. japonica and S. angulatus leaves. leaf disk was used for the test of A. lewisii, and a T. TLC assay. To detect cucurbitacin in cucurbita- kirilowii var. japonica leaf disk was used for the ceous plant leaves, thin layer chromatography test of A. nigripennis. The methanol extract of D. (TLC) assay was conducted. Metcalf et al. (1980) palmatus leaves was dissolved in ethanol. The con- reported that Diabrotica species fed on TLC plate centration of the test solution was adjusted to 1 g contained cucurbitacins at the concentration of ng leaf equivalent/ml. The ethanol solution (0.03 ml) order. TLC assay is efficient for detection of cucur- was applied uniformly to the upper surface of the bitacins because cucurbitacins and impurities are leaf disk. The solvent was removed quickly by air- separated by TLC. We used A. lewisii for cucur- drying. The leaflet treated with ethanol was used as bitacin detection because the beetle was strongly a control. The test was conducted in the same man- stimulated to feed by cucurbitacins and can re- ner described above. spond at ng order of cucurbitacins (Abe et al., HPLC analysis. To quantify cucurbitacin con- 2000); they are also slightly stimulated to feed by tained in eighteen cucurbitaceous plant leaves, methanol extracts on many cucurbitaceous plant high performance liquid chromatography (HPLC) leaves. Twenty microliters of the same sample used analysis was performed by a procedure referred to for HPLC analysis was applied on the TLC plate Halaweish and Tallamy (1993). Methanol extract (Silica gel 60 F254, 0.25 mm thick, 5 10 cm, (mentioned above, 20 mg) was partitioned between Merck) by a micro syringe. The TLC plate was de- chloroform and water (5 ml each). The chloroform veloped by a solvent that consisted of hexane, ethyl layer was removed from the solvent by rotary evap- acetate, methanol, and water at a ratio of 12 : 76 : orator under reduced pressure to dryness (tempera- 4:1, respectively. After development was finished, 164 M. ABE and K. MATSUDA solvent was completely removed by air-drying, and the plate was then used for bioassay. A filter paper (12.5 cm diameter) was placed at the bottom of a petri dish (12 cm diameter), and distilled water (2 ml) was added to maintain humidity. The TLC plate was then put on the filter paper. Ten A. lewissi adults (mixed sex and starved for 24 h) were released in the petri dish and allowed to feed the plate for 24 h, under the same conditions for rear- ing described above. After completion of the test, the plate was checked for feeding marks and the presence of saliva. The presence of cucurbitacins B, E, I, and E-glucoside was evaluated with the Rf value of the standard of the cucurbitacins. Statistical analysis. The amount of fresh leaves fed on and the feeding stimulant index among cucurbitaceous plant species were compared using Kruskal-Wallis test and Scheffe’s F-test. The difference in the amount fed on between control leaves and leaves treated with methanol extract was compared using Mann-Whitney U-test. We performed these statistical analyses using StatView, version 4.5 (1996).

RESULTS Feeding preference for leaves Fig. 1. Feeding preference by three beetle species on leaves of 18 cucurbitaceous plant species. Feeding amount is A. indica fed on most cucurbitaceous plant shown as the weight comsumed (mg, meanSE). Letters indi- species used for the test and preference was in the cate the beetle species as follows: A: A. indica, B: A. lewisii, order of L. sicerariaM. japonicaM. liukiuensis C: A. nigripennis. Different lower case letters indicate signifi- S. angulatusS. eduleC. maximaC. melo leaves. cant difference by Scheffe’s F-test (p0.05). A. indica only minimally fed on G. pentaphyllum, and did not feed at all on D. palmatus, M. charan- bitacins or the methanol extract of T. kirilowii var. tia leaves (Fig. 1A). japonica leaves, but did not feed on the leaves. A. lewisii fed almost solely on L. cylindrica leaves. The beetle hardly fed at all on B. hispida, T. Feeding responses to methanol extracts kirilowii var. japonica, S. angulatus, L. siceraria, A. indica was stimulated to feed by methanol ex- and S. brioniaefolius leaves, and did not feed on tracts from plant leaves on which the beetle fed and the leaves of other plant species (Fig. 1B). intensely chewed on the test filter paper treated The preference of A. nigripennis was in the with the methanol extracts. Conversely, the beetle order of T. kirilowii var. japonicaS. angulatusT. hardly responded to the extracts from D. palmatus, bracteataS. edule leaves. The beetle hardly fed G. pentaphylla, L. cylindrica, and M. charantia on the other plant leaves and did not feed on C. leaves, which the beetle did not prefer (Fig. 2A). lanatus, D. palmatus, M. charantia, and M. liuki- A. lewisii showed intense feeding response to a uensis leaves at all (Fig. 1C). test filter paper treated with extracts from L. cylindrica and L. siceraria leaves. The beetles were Feeding response to D. palmatus leaf treated weakly stimulated to feed by the extracts from C. with cucurbitacin sativus, C. melo, and S. angulatus leaves. No feed- The three beetle species frequently tasted the ing response was observed on other plant extracts surface of D. palmatus leaves treated with cucur- (Fig. 2B). Feeding Preference of Three Aulacophora Leaf Beetle Species 165

Detection of cucurbitacins by HPLC analysis Cucurbitacins B and E-glucoside were detected from C. melo, L. siceraria, and S. angulatus leaves by comparison with the standard sample of cucurbitacins. The recovery of cucurbitacins B and E- glucoside through analysis was 80.1% and 71%, respectively. C. melo leaf contained cucurbitacin E- glucoside, and the concentration was 3.4 mg/g fresh leaves. L. siceraria and S. angulatus leaves contained cucurbitacin B, and the concentration was 4.0 and 4.7 mg/g fresh leaves, respectively.

TLC assay A. lewisii secreted brownish saliva and fed on the TLC plate at the same Rf value as cucurbitacins when cucurbitacins (B, E, E-glucoside, and I: 1 mg each) were applied on the TLC plate. When a TLC plate treated with 0.01 mg of cucurbitacins was provided, A. lewisii did not feed on the plate but salivary secretion (brown spots) was observed on the TLC plate. We interpreted this result as an indica- tion that cucurbitacins can be detected by the TLC method. In 20 ml application of the test sample, salivary secretions were observed on the TLC plate treated with the extracts of L. siceraria and S. an- Fig. 2. Feeding responses by three beetle species to methanol extracts of 18 cucurbitaceous plant species. Feeding gulatus leaves at the same Rf value as cucurbitacin stimulatnt activity is shown as a feeding stimulant index. Let- B and E-glucoside. For the extract from C. melo ters indicate the beetle species as follows: A: A. indica, B: A. leaf, salivary secretion was observed at the same Rf lewisii, C: A. nigripennis. Different lower case letters indicate value as cucurbitacin B (Fig. 3). signiﬁcant difference by Scheffe’s F-test (p0.05).

DISCUSSION A. nigripennis intensely chewed the test ﬁlter paper treated with the extracts from S. angulatus, Our results revealed that there were distinct dif- S. edule, and T. kirilowii var. japonica leaves that ferences in feeding preference among the three were highly preferred by the beetle, while the bee- Aulacophora beetle species. Moreover, feeding tle hardly responded to any other plant extracts preference to the fresh leaves was correlated with (Fig. 2C). the preference for the methanol extracts. For this reason, we conclude that the feeding preference by Feeding response to host plant leaves treated the three Aulacophora beetle species among cucur- with D. palmatus extract bitaceous plants was probably restricted by con- Each beetle species fed on control host leaves stituents in the leaves. HPLC analysis and TLC (A. indica: 12.11.5 mg; A. lewisii: 10.10.6 mg; assay revealed that C. melo, L. siceraria, and S. an- A. nigripennis: 8.71.4 mg) but did not feed on the gulatus leaves contain the cucurbitacins B or E- host leaves treated with the methanol extract from glucoside, which act as feeding stimulants for A. D. palmatus leaves (the difference in the amount indica and A. lewisii, in high amounts. Neverthe- fed on was signiﬁcant between the control and less, A. lewisii fed almost solely on L. cylindrica treated leaves by Mann-Whitney U-test at leaf, and the methanol extracts. C. melo and S. an- P0.01). gulatus leaves possibly contain some feeding deterrent for A. lewisii which reduce the strong feeding stimulant activity by the cucurbitacins because A. 166 M. ABE and K. MATSUDA

Fig. 3. Feeding response of A. lewisii to cucurbitacins separated by TLC. Standard cucurbitacins (B, E, I, and glu.: E-glucoside) were 0.01 mg each on TLC. Abbreviations indicate cucurbitacins and the extract from each plant leaf. Cucs: standard cucurbitacins, Sy: Sicyos angulatus, La: Lagenaria siceraria, Lu: Luffa cylindrica, Cm: Cucumis melo, Cp: Cucurbita maxima, Cs: Cu- cumis sativus, Tr: Trichosanthes rostrata, Tb: Trichosanthes bracteata, Tk: Trichosanthes kirilowii var. japonica. lewisii did not feed on the leaves and was only These facts indicate that A. indica is stimulated to weakly stimulated to feed by the methanol extracts. feed by more plant constituents, and is less affected While A. lewisii showed strong feeding response to by feeding deterrents in the plant leaves than the the methanol extract of L. siceraria in spite of the other two species. A. indica possibly has an ability small feeding amount of the leaf. L. siceraria leaf to accept more constituents in plant leaves and use is thick, with fine hairs on the surface, therefore, A. more plant species as a food than the other two lewisii may be prevented from feeding. It is neces- Aulacophora species do. sary to further investigate whether the hair on L. A. nigripennis preferred both leaves and the siceraria leaf is the feeding deterrent factor for A. methanol extracts of T. kirilowii var. japonica, lewisii. Interestingly, cucurbitacins B, E, I, and E- which is a main host for A. nigripennis, and cucur- glucoside were not detected in L. cylindrica leaves, bitacins were not detected in the leaves. Therefore, which were the most preferred by A. lewisii. These we think that some feeding stimulant other than the facts indicate that L. cylindrica leaves contain not cucurbitacins is present in T. kirilowii var. japonica the four cucurbitacins but feeding stimulants for A. leaves that acts as a feeding stimulant for A. ni- lewisii. Because of a lack of feeding stimulants and gripennis. This is supported by the fact that A. ni- the presence of feeding deterrent, plants other than gripennis was hardly stimulated to feed by cucur- L. cylindrica are probably not used by A. lewisii as bitacin B and was not stimulated by cucurbitacins hosts. E, I, and E-glucoside (Abe et al., 2000). Although Although A. indica, like A. lewisii, is strongly the feeding stimulant is unknown as yet, Saito stimulated to feed by the four cucurbitacins, their (1986) reported that A. nigripennis feed on carna- feeding preferences of the two species contrasted tion (Caryophyllaceae). This fact also indicates that notably in that A. indica fed on various cucurbita- A. nigripennis was probably stimulated to feed by ceous plant leaves and was stimulated to feed by some constituents other than the cucurbitacins. the methanol extracts regardless of the presence or Our results also revealed that to the three beetle absence of the four cucurbitacins. Mishima (1936) species, there are unpalatable plants in 18 plant reported that A. indica adults feed not only on cu- species. Interestingly, L. cylindrica was most pre- curbitaceous plants but also on various other plants ferred by A. lewisii but was not preferred by the among 19 families, including a total of 69 species. other two species. L. cylindrica may contain a spe- Feeding Preference of Three Aulacophora Leaf Beetle Species 167 cific feeding stimulant for A. lewisii. In South-East REFERENCES Asia, Aulacophora atripennis prefers the genus Luffa (Bogwat and Pandey, 1967; Singh and Abe, M. and K. Matsuda (2000) Feeding deterrents from Morey, 1981; Khan and Hajela, 1987). Moreover, Momordica charantia leaves to cucurbitaceous feeding beetle species. Appl. Entomol. Zool. 35: 143–149. M. charantia leaves contain momordicines I and II Abe, M., K. Matsuda and Y. Tamaki (2000) Differences in which are feeding deterrents for A. indica, A. fove- feeding response among three cucurbitaceous feeding icollis, and A. nigripennis (Chandravadana, 1987; leaf beetles to cucurbitacins. Appl. Entomol. Zool. 35: Abe and Matsuda, 2000). However, A. stevensi and 137–142. A. cincta in India use M. charantia as a host (Met- Bogwat, J. K. and S. N. Pandey (1967) Food preference in calf, 1979; Metcalf and Metcalf, 1992). On the Aulacophora spp. Indian J. Entomol. 29: 349–352. Chambliss, O. L. and C. M. Jones (1966) Cucurbitacins: spe- other hand, while D. palmatus is a host of a phy- cific insect attractants in Cucurbitaceae. Science 153: tophagous lady beetle, Epilachna boisduvari 1392–1393. (Nakano and Katakura, 2000), it has not been re- Chandravadana, M. V. (1987) Identification of triterpenoid ported that Aulacophora and other leaf beetle antifeedant of red pumpkin beetles (Aulacophora fove- species use D. palmatus as a host. The three icollis) from Momordica charantia. J. Chem. Ecol. 13: 1689–1694. species of the beetle could not feed on D. palmatus Eben, A., M. E. Barbercheck and S. M. Aluja (1997) Mexi- leaves even when treated with the cucurbitacins or can diabroticite beetles: II. Test for preference of cucurbit methanol extract of host plant leaves. The feeding hosts by Acalymma and Diabrotica spp. Environ. Ento- test using the host plant leaves revealed that the mol. 82: 63–72. methanol extract from D. palmatus leaves con- Ferguson, J. E., E. R. Metcalf, R. L. Metcalf and A. M. Rhodes tained a feeding deterrent. For these reasons, it is (1983) Influence of cucurbitacin content in cotyledons of Cucurbitaceae cultivars upon feeding behavior of dia- apparent that D. palmatus leaves certainly contain broticina beetles (Coleoptera: Chrysomelidae). J. Econ. some strong feeding deterrents to the beetles which Entomol. 76: 47–51. mask the feeding stimulant activity of the cucur- Halaweish, F. T. and D. W. Tallamy (1993) Quantitative de- bitacins. These facts indicate that the food plant termination of cucurbitacins by high performance liquid range among genus Aulacophora is full of variety chromatography and high performance thin layer chromatography. J. Liquid Chromatogr. 16: 497–511. and is restricted by not only the cucurbitacins but Khan, M. Z. and K. P. Hajela (1987) Studies on Aulacophora also the constituents in each plant leaf. foveicollis Lucas (Coleoptera: Chrysomelidae)—food The host plant selection mechanism by Aula- preference and extent of damage. Indian J. Entomol. 49: cophora beetles remains largely unclear. Studies on 457–459. the feeding response of Aulacophora beetle species Kitajima, J. and Y. Tanaka (1989) Studies on the constituents to cucurbitacins revealed the feeding stimulant ac- of trichosanthes root. I. Constituents of roots of Tri- chosanthes kirilowii Maxim. var. japonicum Kitum. tivity of cucurbitacins: A. foveicollis was stimu- Yakugaku Zasshi 109: 250–255 (in Japanese with English lated to feed by cucurbitacin E (Shinha and summary). Krishna, 1969, 1970), A. lewisii and A. coffeae Lavie, D. and E. Glotter (1971) The cucurbitacins, a group strongly responded to cucurbitacin D (Lewis and of tetracyclic triterpenes. Forts. Chem. Organ. Metcalf, 1996), A. indica and A. lewisii were stim- Naturstoffe 29: 306–362. Lewis, P. A. and R. L. Metcalf (1996) Behavior and ecology ulated to feed by cucurbitacins B, E, I, and E-glu- of Old World Luperini beetles of the genus Aulacophora coside (Abe et al., 2000). However, the feeding (Coleoptera: Chrysomelidae). Chemoecology 7: 150– preference of Aulacophora beetle species to the 155. other cucurbitacins is unknown as yet. More than Metcalf, R. L. (1979) Plants, chemicals, and insects: some 20 chemically different cucurbitacins have been aspects of coevolution. Bull. Entomol. Soc. Am. 25: identified from plants (Lavie and Glotter, 1971); 30–35. Metcalf, R. L. and E. R. Metcalf (1992) Diabroticite root- cucurbitacins other than cucurbitacins B, D, E, I, worm beetles. In Plant Kairomones in Insect Ecology and and E-glucoside possibly act as a specific feeding Control. Chapman and Hall, New York, pp. 64–108. stimulants for Aulacophora beetle species. Contin- Metcalf, R. L., A. M. Rhodes, R. A. Metcalf, J. E. Ferguson, ued the study along these themes should gradually E. R. Metcalf and P-y Lu (1982) Cucurbitacin contents clarify host plant selection mechanisms by phy- and Diabroticites (Coleoptera: Chrysomelidae) feeding upon Cucurbita spp. Environ. Entomol. 11: 931–937. tophagous insects. Metcalf, R. L., J. R. Sanborn and M. Rhodes (1980) Cucur- bitacins as kairomones for diabroticite beetles. Proc. 168 M. ABE and K. MATSUDA

Natl. Acad. Sci. USA 77: 3769–3772. Rehm, S. and J. H. Wessels (1957) Bitter principles of Cu- Mishima, R. (1936) Studies on the life history and preven- curbitaceae. VIII. Cucurbitacins in seedlings—occur- tion of Rhaphidopalpa femoralis. Special report of the rence, biochemistry and genetical aspects. J. Sci. Food Nara Prefectural Agricultural Experiment Station 5: Agric. 8: 687–691. 1–170 (in Japanese). Saito, T. (1986) Life history of Aulacophora nigripennis Nakano, S. and H. Katakura (2000) Morphology and biology Motschulsky attacking the carnation and its control. of a phytophagous ladybird beetle, Epilachna pusillanima Shokubutu Boueki 40: 476–479 (in Japanese). (Coleoptera: Coccinellidae) newly recorded on Ishigaki Shinha, A. K. and S. S. Krishna (1969) Feeding of Aula- Island, the Ryukyus. Appl. Entomol. Zool. 34: 189–194. cophora foveicollis on cucurbitacin. J. Econ. Entomol. Nishida, R., H. Fukami, Y. Tanaka, P. Magalhaes, M. 62: 512–513. Yo koyama and A. Blumenschein (1986) Isolation of Shinha, A. K. and S. S. Krishna (1970) Further studies on feeding stimulants of Brazilian leaf beetles (Diabrotica the feeding behavior of Aulacophora foveicollis on cucur- speciosa and Ceratoma arcuata) from the roots of Cer- bitacin. J. Econ. Entomol. 63: 333–334. atosanthes hilariana. Agric. Biol. Chem. 50: 2831– Singh, Z. and R. J. Morey (1981) The feeding preference, 2836. rate of development and fecundity of two pumpkin bee- Rehm, S., P. R. Enslin, A. D. Meeuse and J. H. Wessels (1957) tles on different cucurbitaceous host plants. Indian J. Bitter principles of Cucurbitaceae. VII. The distribution Entomol. 8: 235–244. of bitter principles in this family. J. Sci. Food Agric. 8: StatView (1996) StatView User’s Manual, Ve r sion 4.5. 679–686. Abacus Concepts, Inc., Berkeley, CA.