Journal of Chemical Ecology, Vol. 12, No. 10, 1986
F L A V A N O N E GLYCOSIDES AS OVIPOSITION STIMULANTS IN A PAPILIONID B U T T E R F L Y ,
Papilio protenor
KEIICHI HONDA Seish6 Biological Laboratory 1-3-11 Naka-cho, Odawara 250, Japan (Received September 11, 1985; accepted December 6, 1985)
Abstract--Identification of chemical compounds responsible for the oviposition behavior in a Rutaceae feeder, Papilio protenor demetrius, was undertaken with the epicarp of sour orange (Citrus natsudaidai) which exhibited potent stimulatory activity as did its leaves for egg-laying by the females. The stimulants were present in the hydrosoluble fraction, and the kairomonal activity displayed by the peel was regarded as originating from the synergistic effect of the total chemical complex. One of the active compounds was identified as a flavanone glycoside, naringin (naringenin-7~-neohespefidoside), which, although showing no appreciable effectiveness when bioassayed alone, elicited positive response at the concentration of 0.2 % either when admixed with other unidentified components or provided the females had been conditioned with them in advance. Another flavanone glycoside, hespefidin (hesperetin-7~-mtinoside) that was contained in a trace amount in the peel also had a positive effect comparable to that of nafingin under similar conditions, while their corresponding aglycones were less active or inactive. In contrast, a flavone glycoside, rhoifolin, coexisting in the peel, and some other flavones and flavonols tested as possible candidates for oviposition stimulants were all found entirely ineffective. Key Words--Oviposition stimulant, Papilio protenor, Lepidoptera, Papilionidae, flavanone glycoside, naringin, hesperidin, synergy, Citrus natsudaidai.
INTRODUCTION
A survey of numerous investigations so far conducted on oviposition behavior in many butterfly species coveting major families such as Pieridae, Papilionidae, Nymphalidae, and Danaidae reveals that the oviposition sequence in gen-
1999 0098-0331/86/1000-1999505.00/0 9 1986PlenumPublishingCorporation
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eral consists of the following successive phases: (1) Searching for host plants guided by visual and/or olfactory cues, (2) semirandom or nonrandom (where search image is formed) alighting on plants, (3) drumming on the surface of foliage with foretarsi for the purpose of discrimination and recognition of the host plant, and (4) abdominal curling and probing appropriate ovipositional site with the ovipositor extruded, followed by egg deposition (Chew and Robbins, 1984). Recent work has documented that in the incipient step of oviposition behavior certain butterflies primarily utilize leaf shape to visually distinguish potential larval food plants from manifold vegetation of which the majority would be unsuitable for larval growth and to enhance the frequency at which they encounter the host plants (Benson, 1978; Rausher, 1978; Stanton, 1982). In selecting the ovipositional sites, females of some species appear to be affected also by the color of leaves of host plants (Ilse, 1937; Vaidya, 1969; Saxena and Goyal, 1978) or by the presence of previously laid conspecific eggs or egg mimics on plants (Rothschild and Schoonhoven, 1977; Rausher, 1979; Shapiro, 1981; Williams and Gilbert, 1981). Despite the profound involvement in host seeking of visual stimuli and, in some cases, of volatile chemicals emitted by host plants which attract preovipositional females (Saxena and Goyal, 1978; Feeny et al., 1983), it is well established in Pieridae and Papilionidae that dominant stimuli acting in the final stage of host selection at close range as the cues permitting the females to assess the suitability of the plant they landed on are secondary plant metabolites. These are liberated during drumming, are perceived by foretarsal chemoreceptors and induce oviposition (David and Gardiner, 1962; Nishida, 1977; Ichinos6 and Honda, 1978; Saxena and Goyal, 1978; Rodman and Chew, 1980; Abe et al., 1981; Feeny et al., 1983). The importance in host recognition of tarsi, especially foretarsi, which are endowed with chemotactile sensory receptors, is fully substantiated in several butterfly families, and their external morphology has been examined microscopically (Myers, 1969; Ma and Schoonhoven, 1973; Calvert, 1974; Ichinos6 and Honda, 1978; Stanton, 1979; Feeny et al., 1983). However, insufficient knowledge of the chemical basis for differential response of ovipositing females to various plants has been accumulated (Nishida, 1977; Ichinos6 and Honda, 1978; Abe et al., 1981; Feeny et al., 1983). Only a few compounds have been identified as a trigger for abdominal curling from which egg-laying ensues. Sinigrin (allylglucosinolate) has been reported to stimulate oviposition in Pieris brassicae (David and Gardiner, 1962) and P. rapae (Renwick and Radke, 1983). Recent study with Papilio xuthus (Ohsugi et al., 1985) has revealed that the ovipositional response is elicited by a flavone glycoside, vicenin-2 (6,8-di-C-fl-D-glucopyranosylapigenin), which, however, was effective only when admixed with other, as yet unidentified, components. The present paper reports on the chemical compounds that evoke egg-laying from an oligophagous papilionid butterfly, Papilio protenor, which in na-
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FLAVANONE GLYCOSIDES
ture exclusively feeds on specific plants in the limited range of genera, Citrus (main), Poncirus, Fagara, and Zanthoxylumbelonging to the family Rutaeeae.
METHODS
AND MATERIALS
Insects. Female butterflies of Papilio protenor demetrius were obtained from stock cultures maintained in our laboratory, or collected in the fields in Kanagawa Prefecture. They are of the same population which infests principally Citrus unshiu and C. natsudaidai in nature. Extraction and Fractionation of Active Compounds. The epicarp of C. natsudaidai was extracted with ca. threefold amount of methanol at room temperature for a month. Female butterflies exhibited positive response to the methanol extract, suggesting that the active substance(s) inducing oviposition is present in the extract. The methanolic extract was concentrated in vacuo below 50~ The residue (13.3 g), dissolved in 100 ml of water, was extracted with three 50-ml portions of isobutanol. The aqueous layer (fraction 2, 11.7 g) was subjected to ultrafiltration at a level of tool wt 1000 with a membrane filter (UH-1, T6y6 Roshi Co., Ltd.) to give two fractions: one (0.7 g) containing compounds with a molecular weight of more than 1000 (fraction 3) and the other (11.0 g) composed of smaller molecules (fraction 4). An aliquot of fraction 4 (1.5 g) was further fractionated by reverse-phase chromatography on a ODS column (LRP-2, 45 /zm, Whatman Inc.; 2 cm ID x 30 cm) yielding fractions 5 (1.36 g), 6 (0.03 g), 7 (trace), 8 (0.06 g), and 9 (trace). The column was eluted stepwise with methanol-H20 (20 : 80), methanol, and acetone, successively. The schematic procedure for separation is shown in Figure 1. Authentic Chemicals. Commercially available naringin and hesperidin (Tokyo Kasei Kogyo Co., Ltd.) were recrystallized from water and pyridine, respectively. Naringenin, rutin (Tokyo Kasei Kogyo Co., Ltd.), hesperetin, rhoifolin, apigenin, diosmin, and kaempferol-7-neohesperidoside (Sigma Chemical Co.) were all used without further purification. Bioassayfor Ovipositional Activity. Artificial green plastic leaves, shaped like a compound leaf (25 cm long) consisting of 15 fusiform "foliole" (16 mm long x 11 mm wide), were dipped into aqueous methanol solution of a test sample of a given concentration and air-dried at room temperature to remove methanol. The artificial leaves were remoistened by spraying distilled water on the surface and presented to 3- to 10-day-old fertile females, which, prior to the experiments, had been fed with 10% aqueous sucrose and kept in a transparent plastic cage (25 x 35 cm; height, 15 cm) that permits them free flight. Appraisal of positive response was based on the following criteria: release of the sequential oviposition behavior usually observed in nature that consists of drumming on a leaf with forelegs and subsequent abdominal curling followed by settling of the ovipositor in contact with the underside of the leaf. The fe-
2002
HONDA Epicarp of Citrus natsudaidai Extr. with MeOH Filtration
[
]
Residue
.
Filtrate
I
Conc. in vacuo
Residue
(7.6%)*
Add H20 Extr. with i-BuOH
[
(I) i__-BuOH layer (0.9%)
1
.
(2) Aq. layer (6.7%)
Ultrafiltr. at M.W. 1000
F (3) above I000 (O.4%)
J . (4) telow I000 (6.3%) Column chromatogr on ODS silica gel
(~)~(9) FIG. 1. Schematic procedure for the separation of oviposition stimulants from the epicarp of sour orange (C. natsudaidai). Fractions marked with asterisk were consistently active when bioassayed with 0.2-0.5 % solution. Relative amount of each fraction to the original fresh weight of the epicarp is given in parentheses.
males screened as available for the behavioral test were only those that showed responses negative to distilled water alone and positive to the leaves of C. natsudaidai. Unresponsiveness of the females to the control (water) was carefully confirmed every three trials of sample presentation, for some females appeared to get "hypersensitive" during the experiment, positively responding without any chemical stimuli other than water. The rate of response was expressed by the number of positive responses/number of alighting on the leaves that was accompanied with drumming. NMR Spectroscopy. [13C]NMR spectra were recorded at 67.8 MHz in CD3OD on a JEOL JNM-GX 270 FT-NMR spectrometer by proton-decoupled operation with tetramethylsilane as the internal standard. Temperature, flip angle, pulse repetition time, and the number of data points were 25~ 45 ~ 5 sec and 64 K, respectively. Chemical shifts were represented by 6 unit. Multiplicity of signals determined by means of INEPT technique was abbreviated as follows: singlet (s), doublet (d), triplet (t), quartet (q).
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FLAVANONE GLYCOSIDES
Mass Spectrometry. Fast-atom-bombardment mass spectra (FAB-MS) were measured with a JEOL JMS-DX 300 mass spectrometer. The target was bombarded with 6 KeV Xe atoms employing a mixture of sodium iodide and glycerine as a matrix. High-Pressure Liquid Chromatography (HPLC). Reverse-phase HPLC analyses were carried out with a Hitachi 635S liquid chromatograph equipped with a wavelength-tunable spectrophotometer using a stainless column (4.6 mm ID • 15 cm) packed with Hitachi gel 3056 (ODS silica gel, 5/zm). Mixed solvent composed of M e O H - H 2 0 - A c O H ( 2 0 : 8 0 : 0 . 1 ) was used as an eluent, and the column eluate was monitored at 280 nm. The system was operated at an ambient temperature, and the flow rate was regulated at 1 mt/min. RESULTS As shown in Figure 1, major active compound(s) was present in the hydrosoluble fraction (fraction 2), and found to be a relatively small molecule whose molecular weight was less than 1000 (fraction 4). Further fractionation by reverse-phase column chromatography on ODS revealed that the active principle is composed of more than two components: the one being contained in fraction 5, and the other in fraction 8 (Figure 2). However, the potency of stimulatory effect on oviposition was quite different between these two fractions. Although 1% solution of fraction 8 was effective to a certain extent, its activity drastically diminished at lower concentrations, and eventually gave out
1001
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! 5
o~
y=
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ne
50 100 L Fr5 J_ Fr6 L_ M e O H / H 2 0
[20/80)
0 150 2UU z~U :~UU _L Fr7 _L Fr8 J- Fr9 JVR(ml) I MeOH ---LMe2COJ
FIG. 2. Ovipositional response of P. protenor to fractions 5-9 separated from fraction 4 by reverse-phase (ODS) column chromatography. Three females were tested, and assay concentration of the sample was 1%. The relative weight of each fraction is indicated by a shaded bar, and the response by a closed circle.
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almost completely at a level of 0.2%, whereas fraction 5 of the same concentration still provoked strong response at a rate as much as 100%. Therefore, the principal oviposition stimulant in the peel of C. natsudaidai was considered to be localized in fraction 5. Upon further chemical analysis by thin-layer chromatography (TLC) on silicic acid with methanol, fraction 5 was found to be a mixture of a number of compounds, and each of the four fractions separated retained sufficient activity for inducing egg-laying at a level of 0.2 %, indicating that at least several compounds combine to evoke oviposition behavior in this species. Since every fraction derived from fraction 5, however, seemed to gradually lose its activity during storage, fraction 5 was not pursued further in this study. In contrast, fraction 8, although its stimulatory activity was not high, appeared far less labile, and gave a yellow precipitate when stored in a refrigerator for several days. The crude precipitate was recrystallized twice from hot water and dried at 150~ to afford pale yellow crystals (rap: 172-173~ which, on HPLC analysis, was found to correspond to the major peak (A) in fraction 8 (Figure 3). Compound A was bioassayed for ovipositional response, but showed no appreciable activity at the concentration of 0.2 % and was only slightly active at the level of 0.5 %. However, the stimulatory effect of this substance, if only the females were "conditioned" beforehand with 0.2% solution of fraction 5, markedly increased to an extent adequate to release egg-laying (Table 1). Although a solid explanation for such remarkable enhancement of activity is hard to find at present (this will be discussed later), compound A should be regarded A,B
t'x
o
10
min
Fro. 3. HPLC chromatogram of fraction 8 on a ODS column. A,B: naringin and rhoifolin; C: hesperidin.
FLAVANONE GLYCOSIDES
2005
TABLE 1. OV1POSIT1ON-STIMULATORY ACTIVITY TO Papilio protenor OF NAMPLES DERIVED FROM EPICARP OF Citrus natsudaidai
Response (No. of trials)" Sample
0.1% h
0.2 %
0.5 %
MeOH extract Fraction 5 Compound A Compound A'
0.23 (22) 0.86 (21) 0 (24) 0 (20)
1.00 (24) 1.00 (21) 0 (22) 0.81 (21)
1.00 (20) 1.00 (22) 0.12 (26) 0.92 (26)
"Twelve females were tested. bPercent indicates concentration of sample. "The femaleswere conditioned with fraction 5 (0.2%) prior to the experiments. Unresponsiveness to the control (water alone) was carefully confirmedafter conditioning to rule out the possibility of a direct effect of fraction 5. as one of the oviposition stimulants. Therefore, an attempt was made at structural elucidation of compound A. The aqueous solution of compound A turned reddish-purple when treated with Mg-HC1, thereby adducing the possibility of the compound being a sort of flavonoid. The result of elemental analysis, combined with FAB-MS information [(M + H) + at m/z 581 and (M + Na) + at m/z 6031, determined the molecular formula of compound A to be C 2 7 H 3 2 0 1 4 . More informative data were obtained by CMR measurement giving the following signals, 6: 197.8(s), 165.9(s), 164.2(s), 164.0(s), 158.4(s), 130.3(s), 130.2(s), 128.7(d), 128.6(d), ll6.0(d), 104.5(s), 102.1(d), 99.0(d), 97.5(d), 96.4(d), 80.3(d), 78.9(d), 78.7(d), 78.5(d), 77.7(d), 73.6(d), 71.9(d), 70.9(d), 69.7(d), 62.0(t), 43.9(t), 43.6(t), 18.2(q), which were suggestive of the presence of one carbonyl carbon, no less than 10 aromatic carbons, and at least 10 carbons that may be assigned to the sugar moiety. Compound A was thus assumed to be a flavonoid glycoside, possibly a flavanone glycoside among others. On the other hand, CMR spectrum of the acid hydrolysis (5% aq. HC1) product (pale yellow crystals soluble in ethyl acetate) of compound A, which was regarded as its aglycone, coincided well with that of authentic naringenin. Furthermore, the resonance at 18.2 ppm assignable to a methyl group was indicative of the presence of rhamnose in the sugar residue. This spectral evidence suggests that compound A is likely to be naringenin-7-rhamnoglucoside, probably either naringin or narirutin. Good agreement in melting point and spectral data of authentic naringin with those of compound A confirmed the identity of the two. Compound A was thus determined to be naringin (naringenin-7fl-neohesperidoside, Figure 4). HPLC analysis and CMR measurement of the crude crystals separated from fraction 8 revealed that fraction 8 also contains a small amount of rhoifolin
2006
HONDA ~
~",,~,-OCH 3 I II
O O-CH2 H CH? O~ .o
&-lo.
HOjr--O0 ~-~ OHOH
Naringin
O
o.
OH
o
Hesperidin
FIG. 4. Chemical structures of flavanone glycosides appraised as active for egg-laying by P. protenor. Naringin: naringenin-7fl-neohesperidoside; Hesperidin: hesperetin-7/3rutinoside. (apigenin-7fi-neohesperidoside, peak B in Figure 3), which is superimposed on peak A. In addition, the existence of a trace of hesperidin (hesperetin-7fi-mtinoside, Figure 4) in fraction 8 was also presumed by HPLC retention data (peak C in Figure 3). Naringin, hesperidin, rhoifolin (the former two being flavanone glycosides, and the latter, a flavone glycoside), and other several flavonoid compounds available as possible candidates for oviposition stimulants were then bioassayed. The results are summarized in Table 2. Unless the females were conditioned with fraction 5 prior to the experiment, naringin was virtually inactive when assayed alone, while pretreatment with or coexistence of fraction 5 remarkably promoted its activity, and eventually naringin turned out active. As all the other substances tested exerted little or no stimulatory action by themselves, their activity was evaluated after similar conditioning. Of these compounds, only flavanones such as naringin, hesperidin, and hesperetin, to which females evidently displayed a positive response, were appraised as active. In contrast, flavones and flavonols were entirely inactive, although limited solubility of some compounds precluded examining their dose-response correlation. As for naringin and hesperidin, the minimal concentration necessary for eliciting ovipositional response by females was estimated equally at ca. 0.2%. The latter, however, appears a little more active since the effectiveness of 0.1% solution of hesperidin was somewhat higher as compared with that of naringin, and besides, its aglycone, hesperetin, exhibited unequivocal ovipositionat activity despite the inertness of naringenin (aglycone of naringin). DISCUSSION
The present results are congruent with previous work by Ichinos6 and Honda (1978) in that egg-laying of Papilio protenor is stimulated by watersoluble substances in Citrus plants and further demonstrate definitely that the
OH OH OMe OMe OH OH OMe OH OH
I I 1 II II II
11 II
R1
I
Type
oT. no
H OH
H OH OH H H OH
H
R2
I
OH Ru
H H H
R3 a
R2
Ne OH
OH Ru OH Ne OH Ru
Ne
R4"
R4",],~O
0.05 0.04 0 0.19 0 0.09
(21) (24) (24) (21) (20) (22)
0 (22)
0.1%"
(25) (25) (24) (25) e (23) (29) (25) g (31) u 0 (23) 0 (20)
0.88 0.84 0 0.80 0.04 0.14 0 0.03
0.08 (26)
0.2%
Response (No. of trials)/'
R2
aRm Rutinose, Ne: Neohesperidose. bRecorded using 12 females that were preconditioned with fraction 5 (0.2%), unless otherwise noted. c Percent indicates concentration of sample. dUnconditioned. eCompounds whose presence in the epicarp of Citrus natsudaidai was confinned or presumed. IFraction 5 elicited no appreciable response at 0.05 %. gBecause of the scanty solubility, some portions of crystals remained indissolved.
Naringind, ," ] Naringin a a n d [ Fr.5 (0.05 %)(J Naringin Naringenin Hesperidin ~" Hesperetin Rhoifolin ~ Apigenin Diosmin Kaempferol-7 neohespeddoside Rutin
Compound
R4
TABLE 2. OVIPOSITIONAL RESPONSE OF Papilio protenor TO FLAVONOID COMPOUNDS
(30) (27) (20) (25) g (23) (24) g (25) ,e (22) g 0 (20) 0 (25)
0.93 0.96 0.15 0.84 0.83 0.13 0.04 0
0.13 (24)
0.5%
t~ o ..q
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HONDA
kairomonal activity for oviposition displayed by the peel of C. natsudaidai is the result of synergistic interaction among manifold chemical compounds. Similar Synergy of chemicals involved in host selection has been reported for the cabbage butterfly, Pieris rapae, where sinigrin, although it stimulates oviposition, does not act alone to mediate discrimination among potential host plants, and combination with other water-soluble compounds evoked more potent ovipositional response (Renwick and Radke, 1983). A recent study with a swallowtail butterfly, Papilio xuthus, has also dealt with an analogous topic where vicenin-2 (flavone glycoside), which was inactive alone, proved active when admixed with another water-soluble fraction (Ohsugi et al., 1985). The stimulatory action of naringin and hesperidinon egg deposition by the females appears to prevail only to a limited extent and to be subsidiary, because they neither exhibited any appreciable activity when assayed alone nor played an essential role in oviposition as evidenced by the fact that fraction 5, devoid of the two compounds, did suffice singly to elicit ovipositional response. Accordingly, such flavanone glycosides are not principal determinants of oviposition; nevertheless, either compound should be regarded as factors conducive to the synergy. Although it is difficult to put an explicit interpretation on the enhanced response by ovipositing females to flavanones caused by pretreatment with fraction 5, a possible reason is that this phenomenon was due to "memory effect" brought about by a trace of residual active substances adsorbed on the tarsal chemoreceptors of forelegs. In fact, repetitive washing of foretarsi with water resulted in much reduced response to flavanones. Naringin and hesperidin are not specific to Citrus plants (although not being widespread in plants); however, most Citrus plants have been reported to be excellent producers of these compounds, which are contained not only in fruits but also in leaves (Hattori et al., 1952; Nakabayashi, 1961; Nishiura et al., 1969; Namba et al., 1985). In this respect, it is very likely that Papilio protenor, one of Citrus feeders, has evolutionarily come to rely not on ubiquitous flavonoids such as mtin (flavonol) but on certain specified flavanones in discriminating more precisely its host plants out of a diversity of unacceptable plants. Some Citrus plants are also known to yield several flavones as minor flavonoid constituents. P. protenor, however, made little or no response to such flavone glycosides as rhoifolin and diosmin, the former being frequently encountered in Citrus plants (Hattori et al., 1952; Nakabayashi, 1961; Nishiura et al., 1969). Therefore, flavones in general appear essentially inactive to P. protenor, whereas a sort of flavone glycoside, vicenin-2, has been reported to be active to a related species, P. xuthus (Ohsugi et al., 1985). Feeny et al. (1983) have shown that for P. polyxenes, a umbellifer-feeding swallowtail, naringin and hesperidin were ineffective altogether, while chlorogenic acid and apigenin-7-glucoside (flavone) were slightly active. Such disparity may partly reflect interspecific difference in host preference.
FLAVANONE GLYCOSIDES
2009
In P. p r o t e n o r , all o f t h e c o m p o n e n t s p r e s e n t in a c t i v e f r a c t i o n s s e e m n o t indispensable for inducing oviposition; however, the whole complex of constituents should form the chemical basis for host recognition and preference. T o p o s s e s s a c o m p r e h e n s i v e r e s p o n s e s p e c t r u m to o r a g a i n s t a v a r i e t y o f c o m p o u n d s , n o t r e l y i n g o n a n y s i n g l e s t i m u l a n t , m a y s e r v e to e n s u r e c o r r e c t a n d strict d i s c r i m i n a t i o n o f h o s t p l a n t s b y m o t h e r b u t t e r f l i e s .
REFERENCES ABE, .H., TERAMOTO, Y., and ICHINOS~, T. 1981. Relationship between host plant ranges of the three papilionid butterflies and oviposition-inducing contact chemicals in their host plants. Appl. Entomol. Zool. 16:493-496. BENSON, W.W. 1978. Resource partitioning in passion vine butterflies. Evolution 32:493-518. CALVERT, W.H. 1974. The external morphology of foretarsal receptors involved with host discrimination by the nymphalid butterfly, Chlosyne lacinia. Ann. Entomol. Soc. Am. 67:853-856. CHEW, F.S., and ROBB1NS,R.K. 1984. Egg-laying in butterflies, pp. 65-79, in R.I. Vane-Wright, and P.R. Ackery (eds.). The Biology of Butterflies. Academic Press, New York. 429 pp. DAVID, W.A.L., and GARDINER, B.O.C. 1962. Oviposition and hatching of the eggs of Pieris brassicae in a laboratory culture. Bull. Entomol. Res. 53:91-109. FEENY, P., ROSENBERRY, L., and CARTER, M. 1983. Chemical aspects of oviposition behavior in butterflies, pp. 27-76, in S. Ahmad (ed.). Herbivorous Insects: Host-Seeking Behavior and Mechanisms. Academic Press, New York. 257 pp. HATTORI,S., SHIMOKORIYAMA,M., and KANAO, M. 1952. Studies on flavanone glycosides. I v. The glycosides of ripe fruit peel and flower petals of Citrus aurantium L. J. Am. Chem. SJc. 74:3614-3615. ICH1NOSE, T., and HONDA, H. 1978. Ovipositional behavior of Papilio protenor demetrius f AMER and the factors involved in its host plants. Appl. Entomol. Zool. 13:103-114. ILSE, D. 1937. New observations on responses to colors in egg-laying butterflies. Nature 140:544545. MA, W.C., and SCHOONHOVEN, L.M. 1973. Tarsal contact chemosensory hairs of the large white butterfly, Pieris brassicae and their possible r61e in oviposition behaviour. Entomol. Exp. Appl. 16:343-357. MYERS, J. 1969. Distribution of foodplant chemoreceptors on the female Florida queen butterfly, Danaus gilippus berenice (Nymphalidae). J. Lepid. Soc. 23:196-198. NAKABAYASHI, T. 1961. Studies on the Citrus flavonoids. Part VII. Flavonoid glycosides in the species of sour orange. Nippon Nogei Kagaku Kaishi 35:945-949 (in Japanese). NAMBA, T., ARAK~, I., MmAGE, M., and HATTORI, M. 1985. Fundamental studies on the evaluation of crude drugs. VIII. Monthly variations in anatomical characteristics and chemical components of the dried fruit peels of Citrus unshiu, C. aurantium, and C. natsudaidai. Shoyakugaku Zasshi 39:52-62. NISHIDA, R. 1977. Oviposition stimulants of some papilionid butterflies contained in their host plants. Botyu-Kagaku 42:133-140 (in Japanese). N1SHIURA, M., ESAKI, S., and KAMIYA, S. 1969. Flavonoids in Citrus and related genera. Part I. Distribution of flavonoid glycosides in Citrus and Poncirus. Agric. Biol. Chem. 33:11091118. OHSUGI, T., NISH1DA, R., and FUKAMI, H. 1985. Oviposition stimulant of Papilio xuthus, a Citrusfeeding swallowtail butterfly. Agric. Biol. Chem. 49:1897-1900. RAUSHER, M.D. 1978. Search image for leaf shape in a butterfly. Science 200:1071-1073. RAUSHER, M.D. 1979. Egg recognition: Its advantage to a butterfly. Anita. Behav. 27:1034-1040.
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RENWICK, J.A.A., and RADKE,C.D. 1983. Chemical recognition of host plants for oviposition by the cabbage butterfly, Pieris rapae (Lepidoptera: Pieddae). Environ. Entomol. 12:446-450. RODMAN, J.E., and CHEW, F.S. 1980. Phytochemical correlates of herbivory in a community of native and naturalized Cmciferae. Biochem. Syst. Ecol. 8:43-50. ROTHSCHILD, M., and SCHOONHOVEN,L.M. 1977. Assessment of egg load by Pieris brassicae. Nature 266:352-355. SAXENA,K.N., and GOYAL,S. 1978. Host-plant relations of the citrus butterfly Papilio demoleus L.: Orientational and ovipositional responses. Entomol. Exp. Appl. 24:1-10. SHAPIRO, A.M. 1981. Egg-mimics of Streptanthus (Cruciferae) deter oviposition by Pieris sisymbrii. Oecologia 48:142 143. STANTON, M.L. 1979. The role of chemotactile stimuli in the oviposition preferences of Colias butterflies. Oecologia 39:79-91. STANTON,M.L. 1982. Searching in a patchy environment: Food plant selection by Colias p. eriphyle butterflies. Ecology 63: 839-853. VAIDYA,V.G. 1969. investigations of the role of visual stimuli in egg-laying and resting behavior of Papilio demoleus L. (Papilionidae, Lepidoptera). Anita. Behav. 17:350-355. WILLIAMS,K.S., and GILBERT,L.E. 1981. Insects as selective agents on plant vegetative morphology: Egg mimicry reduces egg laying by butterflies. Science 212:467-469.
F L A V A N O N E GLYCOSIDES AS OVIPOSITION STIMULANTS IN A PAPILIONID B U T T E R F L Y ,
Papilio protenor
KEIICHI HONDA Seish6 Biological Laboratory 1-3-11 Naka-cho, Odawara 250, Japan (Received September 11, 1985; accepted December 6, 1985)
Abstract--Identification of chemical compounds responsible for the oviposition behavior in a Rutaceae feeder, Papilio protenor demetrius, was undertaken with the epicarp of sour orange (Citrus natsudaidai) which exhibited potent stimulatory activity as did its leaves for egg-laying by the females. The stimulants were present in the hydrosoluble fraction, and the kairomonal activity displayed by the peel was regarded as originating from the synergistic effect of the total chemical complex. One of the active compounds was identified as a flavanone glycoside, naringin (naringenin-7~-neohespefidoside), which, although showing no appreciable effectiveness when bioassayed alone, elicited positive response at the concentration of 0.2 % either when admixed with other unidentified components or provided the females had been conditioned with them in advance. Another flavanone glycoside, hespefidin (hesperetin-7~-mtinoside) that was contained in a trace amount in the peel also had a positive effect comparable to that of nafingin under similar conditions, while their corresponding aglycones were less active or inactive. In contrast, a flavone glycoside, rhoifolin, coexisting in the peel, and some other flavones and flavonols tested as possible candidates for oviposition stimulants were all found entirely ineffective. Key Words--Oviposition stimulant, Papilio protenor, Lepidoptera, Papilionidae, flavanone glycoside, naringin, hesperidin, synergy, Citrus natsudaidai.
INTRODUCTION
A survey of numerous investigations so far conducted on oviposition behavior in many butterfly species coveting major families such as Pieridae, Papilionidae, Nymphalidae, and Danaidae reveals that the oviposition sequence in gen-
1999 0098-0331/86/1000-1999505.00/0 9 1986PlenumPublishingCorporation
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eral consists of the following successive phases: (1) Searching for host plants guided by visual and/or olfactory cues, (2) semirandom or nonrandom (where search image is formed) alighting on plants, (3) drumming on the surface of foliage with foretarsi for the purpose of discrimination and recognition of the host plant, and (4) abdominal curling and probing appropriate ovipositional site with the ovipositor extruded, followed by egg deposition (Chew and Robbins, 1984). Recent work has documented that in the incipient step of oviposition behavior certain butterflies primarily utilize leaf shape to visually distinguish potential larval food plants from manifold vegetation of which the majority would be unsuitable for larval growth and to enhance the frequency at which they encounter the host plants (Benson, 1978; Rausher, 1978; Stanton, 1982). In selecting the ovipositional sites, females of some species appear to be affected also by the color of leaves of host plants (Ilse, 1937; Vaidya, 1969; Saxena and Goyal, 1978) or by the presence of previously laid conspecific eggs or egg mimics on plants (Rothschild and Schoonhoven, 1977; Rausher, 1979; Shapiro, 1981; Williams and Gilbert, 1981). Despite the profound involvement in host seeking of visual stimuli and, in some cases, of volatile chemicals emitted by host plants which attract preovipositional females (Saxena and Goyal, 1978; Feeny et al., 1983), it is well established in Pieridae and Papilionidae that dominant stimuli acting in the final stage of host selection at close range as the cues permitting the females to assess the suitability of the plant they landed on are secondary plant metabolites. These are liberated during drumming, are perceived by foretarsal chemoreceptors and induce oviposition (David and Gardiner, 1962; Nishida, 1977; Ichinos6 and Honda, 1978; Saxena and Goyal, 1978; Rodman and Chew, 1980; Abe et al., 1981; Feeny et al., 1983). The importance in host recognition of tarsi, especially foretarsi, which are endowed with chemotactile sensory receptors, is fully substantiated in several butterfly families, and their external morphology has been examined microscopically (Myers, 1969; Ma and Schoonhoven, 1973; Calvert, 1974; Ichinos6 and Honda, 1978; Stanton, 1979; Feeny et al., 1983). However, insufficient knowledge of the chemical basis for differential response of ovipositing females to various plants has been accumulated (Nishida, 1977; Ichinos6 and Honda, 1978; Abe et al., 1981; Feeny et al., 1983). Only a few compounds have been identified as a trigger for abdominal curling from which egg-laying ensues. Sinigrin (allylglucosinolate) has been reported to stimulate oviposition in Pieris brassicae (David and Gardiner, 1962) and P. rapae (Renwick and Radke, 1983). Recent study with Papilio xuthus (Ohsugi et al., 1985) has revealed that the ovipositional response is elicited by a flavone glycoside, vicenin-2 (6,8-di-C-fl-D-glucopyranosylapigenin), which, however, was effective only when admixed with other, as yet unidentified, components. The present paper reports on the chemical compounds that evoke egg-laying from an oligophagous papilionid butterfly, Papilio protenor, which in na-
2001
FLAVANONE GLYCOSIDES
ture exclusively feeds on specific plants in the limited range of genera, Citrus (main), Poncirus, Fagara, and Zanthoxylumbelonging to the family Rutaeeae.
METHODS
AND MATERIALS
Insects. Female butterflies of Papilio protenor demetrius were obtained from stock cultures maintained in our laboratory, or collected in the fields in Kanagawa Prefecture. They are of the same population which infests principally Citrus unshiu and C. natsudaidai in nature. Extraction and Fractionation of Active Compounds. The epicarp of C. natsudaidai was extracted with ca. threefold amount of methanol at room temperature for a month. Female butterflies exhibited positive response to the methanol extract, suggesting that the active substance(s) inducing oviposition is present in the extract. The methanolic extract was concentrated in vacuo below 50~ The residue (13.3 g), dissolved in 100 ml of water, was extracted with three 50-ml portions of isobutanol. The aqueous layer (fraction 2, 11.7 g) was subjected to ultrafiltration at a level of tool wt 1000 with a membrane filter (UH-1, T6y6 Roshi Co., Ltd.) to give two fractions: one (0.7 g) containing compounds with a molecular weight of more than 1000 (fraction 3) and the other (11.0 g) composed of smaller molecules (fraction 4). An aliquot of fraction 4 (1.5 g) was further fractionated by reverse-phase chromatography on a ODS column (LRP-2, 45 /zm, Whatman Inc.; 2 cm ID x 30 cm) yielding fractions 5 (1.36 g), 6 (0.03 g), 7 (trace), 8 (0.06 g), and 9 (trace). The column was eluted stepwise with methanol-H20 (20 : 80), methanol, and acetone, successively. The schematic procedure for separation is shown in Figure 1. Authentic Chemicals. Commercially available naringin and hesperidin (Tokyo Kasei Kogyo Co., Ltd.) were recrystallized from water and pyridine, respectively. Naringenin, rutin (Tokyo Kasei Kogyo Co., Ltd.), hesperetin, rhoifolin, apigenin, diosmin, and kaempferol-7-neohesperidoside (Sigma Chemical Co.) were all used without further purification. Bioassayfor Ovipositional Activity. Artificial green plastic leaves, shaped like a compound leaf (25 cm long) consisting of 15 fusiform "foliole" (16 mm long x 11 mm wide), were dipped into aqueous methanol solution of a test sample of a given concentration and air-dried at room temperature to remove methanol. The artificial leaves were remoistened by spraying distilled water on the surface and presented to 3- to 10-day-old fertile females, which, prior to the experiments, had been fed with 10% aqueous sucrose and kept in a transparent plastic cage (25 x 35 cm; height, 15 cm) that permits them free flight. Appraisal of positive response was based on the following criteria: release of the sequential oviposition behavior usually observed in nature that consists of drumming on a leaf with forelegs and subsequent abdominal curling followed by settling of the ovipositor in contact with the underside of the leaf. The fe-
2002
HONDA Epicarp of Citrus natsudaidai Extr. with MeOH Filtration
[
]
Residue
.
Filtrate
I
Conc. in vacuo
Residue
(7.6%)*
Add H20 Extr. with i-BuOH
[
(I) i__-BuOH layer (0.9%)
1
.
(2) Aq. layer (6.7%)
Ultrafiltr. at M.W. 1000
F (3) above I000 (O.4%)
J . (4) telow I000 (6.3%) Column chromatogr on ODS silica gel
(~)~(9) FIG. 1. Schematic procedure for the separation of oviposition stimulants from the epicarp of sour orange (C. natsudaidai). Fractions marked with asterisk were consistently active when bioassayed with 0.2-0.5 % solution. Relative amount of each fraction to the original fresh weight of the epicarp is given in parentheses.
males screened as available for the behavioral test were only those that showed responses negative to distilled water alone and positive to the leaves of C. natsudaidai. Unresponsiveness of the females to the control (water) was carefully confirmed every three trials of sample presentation, for some females appeared to get "hypersensitive" during the experiment, positively responding without any chemical stimuli other than water. The rate of response was expressed by the number of positive responses/number of alighting on the leaves that was accompanied with drumming. NMR Spectroscopy. [13C]NMR spectra were recorded at 67.8 MHz in CD3OD on a JEOL JNM-GX 270 FT-NMR spectrometer by proton-decoupled operation with tetramethylsilane as the internal standard. Temperature, flip angle, pulse repetition time, and the number of data points were 25~ 45 ~ 5 sec and 64 K, respectively. Chemical shifts were represented by 6 unit. Multiplicity of signals determined by means of INEPT technique was abbreviated as follows: singlet (s), doublet (d), triplet (t), quartet (q).
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FLAVANONE GLYCOSIDES
Mass Spectrometry. Fast-atom-bombardment mass spectra (FAB-MS) were measured with a JEOL JMS-DX 300 mass spectrometer. The target was bombarded with 6 KeV Xe atoms employing a mixture of sodium iodide and glycerine as a matrix. High-Pressure Liquid Chromatography (HPLC). Reverse-phase HPLC analyses were carried out with a Hitachi 635S liquid chromatograph equipped with a wavelength-tunable spectrophotometer using a stainless column (4.6 mm ID • 15 cm) packed with Hitachi gel 3056 (ODS silica gel, 5/zm). Mixed solvent composed of M e O H - H 2 0 - A c O H ( 2 0 : 8 0 : 0 . 1 ) was used as an eluent, and the column eluate was monitored at 280 nm. The system was operated at an ambient temperature, and the flow rate was regulated at 1 mt/min. RESULTS As shown in Figure 1, major active compound(s) was present in the hydrosoluble fraction (fraction 2), and found to be a relatively small molecule whose molecular weight was less than 1000 (fraction 4). Further fractionation by reverse-phase column chromatography on ODS revealed that the active principle is composed of more than two components: the one being contained in fraction 5, and the other in fraction 8 (Figure 2). However, the potency of stimulatory effect on oviposition was quite different between these two fractions. Although 1% solution of fraction 8 was effective to a certain extent, its activity drastically diminished at lower concentrations, and eventually gave out
1001
'~
! 5
o~
y=
,0 e',,
ne
50 100 L Fr5 J_ Fr6 L_ M e O H / H 2 0
[20/80)
0 150 2UU z~U :~UU _L Fr7 _L Fr8 J- Fr9 JVR(ml) I MeOH ---LMe2COJ
FIG. 2. Ovipositional response of P. protenor to fractions 5-9 separated from fraction 4 by reverse-phase (ODS) column chromatography. Three females were tested, and assay concentration of the sample was 1%. The relative weight of each fraction is indicated by a shaded bar, and the response by a closed circle.
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almost completely at a level of 0.2%, whereas fraction 5 of the same concentration still provoked strong response at a rate as much as 100%. Therefore, the principal oviposition stimulant in the peel of C. natsudaidai was considered to be localized in fraction 5. Upon further chemical analysis by thin-layer chromatography (TLC) on silicic acid with methanol, fraction 5 was found to be a mixture of a number of compounds, and each of the four fractions separated retained sufficient activity for inducing egg-laying at a level of 0.2 %, indicating that at least several compounds combine to evoke oviposition behavior in this species. Since every fraction derived from fraction 5, however, seemed to gradually lose its activity during storage, fraction 5 was not pursued further in this study. In contrast, fraction 8, although its stimulatory activity was not high, appeared far less labile, and gave a yellow precipitate when stored in a refrigerator for several days. The crude precipitate was recrystallized twice from hot water and dried at 150~ to afford pale yellow crystals (rap: 172-173~ which, on HPLC analysis, was found to correspond to the major peak (A) in fraction 8 (Figure 3). Compound A was bioassayed for ovipositional response, but showed no appreciable activity at the concentration of 0.2 % and was only slightly active at the level of 0.5 %. However, the stimulatory effect of this substance, if only the females were "conditioned" beforehand with 0.2% solution of fraction 5, markedly increased to an extent adequate to release egg-laying (Table 1). Although a solid explanation for such remarkable enhancement of activity is hard to find at present (this will be discussed later), compound A should be regarded A,B
t'x
o
10
min
Fro. 3. HPLC chromatogram of fraction 8 on a ODS column. A,B: naringin and rhoifolin; C: hesperidin.
FLAVANONE GLYCOSIDES
2005
TABLE 1. OV1POSIT1ON-STIMULATORY ACTIVITY TO Papilio protenor OF NAMPLES DERIVED FROM EPICARP OF Citrus natsudaidai
Response (No. of trials)" Sample
0.1% h
0.2 %
0.5 %
MeOH extract Fraction 5 Compound A Compound A'
0.23 (22) 0.86 (21) 0 (24) 0 (20)
1.00 (24) 1.00 (21) 0 (22) 0.81 (21)
1.00 (20) 1.00 (22) 0.12 (26) 0.92 (26)
"Twelve females were tested. bPercent indicates concentration of sample. "The femaleswere conditioned with fraction 5 (0.2%) prior to the experiments. Unresponsiveness to the control (water alone) was carefully confirmedafter conditioning to rule out the possibility of a direct effect of fraction 5. as one of the oviposition stimulants. Therefore, an attempt was made at structural elucidation of compound A. The aqueous solution of compound A turned reddish-purple when treated with Mg-HC1, thereby adducing the possibility of the compound being a sort of flavonoid. The result of elemental analysis, combined with FAB-MS information [(M + H) + at m/z 581 and (M + Na) + at m/z 6031, determined the molecular formula of compound A to be C 2 7 H 3 2 0 1 4 . More informative data were obtained by CMR measurement giving the following signals, 6: 197.8(s), 165.9(s), 164.2(s), 164.0(s), 158.4(s), 130.3(s), 130.2(s), 128.7(d), 128.6(d), ll6.0(d), 104.5(s), 102.1(d), 99.0(d), 97.5(d), 96.4(d), 80.3(d), 78.9(d), 78.7(d), 78.5(d), 77.7(d), 73.6(d), 71.9(d), 70.9(d), 69.7(d), 62.0(t), 43.9(t), 43.6(t), 18.2(q), which were suggestive of the presence of one carbonyl carbon, no less than 10 aromatic carbons, and at least 10 carbons that may be assigned to the sugar moiety. Compound A was thus assumed to be a flavonoid glycoside, possibly a flavanone glycoside among others. On the other hand, CMR spectrum of the acid hydrolysis (5% aq. HC1) product (pale yellow crystals soluble in ethyl acetate) of compound A, which was regarded as its aglycone, coincided well with that of authentic naringenin. Furthermore, the resonance at 18.2 ppm assignable to a methyl group was indicative of the presence of rhamnose in the sugar residue. This spectral evidence suggests that compound A is likely to be naringenin-7-rhamnoglucoside, probably either naringin or narirutin. Good agreement in melting point and spectral data of authentic naringin with those of compound A confirmed the identity of the two. Compound A was thus determined to be naringin (naringenin-7fl-neohesperidoside, Figure 4). HPLC analysis and CMR measurement of the crude crystals separated from fraction 8 revealed that fraction 8 also contains a small amount of rhoifolin
2006
HONDA ~
~",,~,-OCH 3 I II
O O-CH2 H CH? O~ .o
&-lo.
HOjr--O0 ~-~ OHOH
Naringin
O
o.
OH
o
Hesperidin
FIG. 4. Chemical structures of flavanone glycosides appraised as active for egg-laying by P. protenor. Naringin: naringenin-7fl-neohesperidoside; Hesperidin: hesperetin-7/3rutinoside. (apigenin-7fi-neohesperidoside, peak B in Figure 3), which is superimposed on peak A. In addition, the existence of a trace of hesperidin (hesperetin-7fi-mtinoside, Figure 4) in fraction 8 was also presumed by HPLC retention data (peak C in Figure 3). Naringin, hesperidin, rhoifolin (the former two being flavanone glycosides, and the latter, a flavone glycoside), and other several flavonoid compounds available as possible candidates for oviposition stimulants were then bioassayed. The results are summarized in Table 2. Unless the females were conditioned with fraction 5 prior to the experiment, naringin was virtually inactive when assayed alone, while pretreatment with or coexistence of fraction 5 remarkably promoted its activity, and eventually naringin turned out active. As all the other substances tested exerted little or no stimulatory action by themselves, their activity was evaluated after similar conditioning. Of these compounds, only flavanones such as naringin, hesperidin, and hesperetin, to which females evidently displayed a positive response, were appraised as active. In contrast, flavones and flavonols were entirely inactive, although limited solubility of some compounds precluded examining their dose-response correlation. As for naringin and hesperidin, the minimal concentration necessary for eliciting ovipositional response by females was estimated equally at ca. 0.2%. The latter, however, appears a little more active since the effectiveness of 0.1% solution of hesperidin was somewhat higher as compared with that of naringin, and besides, its aglycone, hesperetin, exhibited unequivocal ovipositionat activity despite the inertness of naringenin (aglycone of naringin). DISCUSSION
The present results are congruent with previous work by Ichinos6 and Honda (1978) in that egg-laying of Papilio protenor is stimulated by watersoluble substances in Citrus plants and further demonstrate definitely that the
OH OH OMe OMe OH OH OMe OH OH
I I 1 II II II
11 II
R1
I
Type
oT. no
H OH
H OH OH H H OH
H
R2
I
OH Ru
H H H
R3 a
R2
Ne OH
OH Ru OH Ne OH Ru
Ne
R4"
R4",],~O
0.05 0.04 0 0.19 0 0.09
(21) (24) (24) (21) (20) (22)
0 (22)
0.1%"
(25) (25) (24) (25) e (23) (29) (25) g (31) u 0 (23) 0 (20)
0.88 0.84 0 0.80 0.04 0.14 0 0.03
0.08 (26)
0.2%
Response (No. of trials)/'
R2
aRm Rutinose, Ne: Neohesperidose. bRecorded using 12 females that were preconditioned with fraction 5 (0.2%), unless otherwise noted. c Percent indicates concentration of sample. dUnconditioned. eCompounds whose presence in the epicarp of Citrus natsudaidai was confinned or presumed. IFraction 5 elicited no appreciable response at 0.05 %. gBecause of the scanty solubility, some portions of crystals remained indissolved.
Naringind, ," ] Naringin a a n d [ Fr.5 (0.05 %)(J Naringin Naringenin Hesperidin ~" Hesperetin Rhoifolin ~ Apigenin Diosmin Kaempferol-7 neohespeddoside Rutin
Compound
R4
TABLE 2. OVIPOSITIONAL RESPONSE OF Papilio protenor TO FLAVONOID COMPOUNDS
(30) (27) (20) (25) g (23) (24) g (25) ,e (22) g 0 (20) 0 (25)
0.93 0.96 0.15 0.84 0.83 0.13 0.04 0
0.13 (24)
0.5%
t~ o ..q
2008
HONDA
kairomonal activity for oviposition displayed by the peel of C. natsudaidai is the result of synergistic interaction among manifold chemical compounds. Similar Synergy of chemicals involved in host selection has been reported for the cabbage butterfly, Pieris rapae, where sinigrin, although it stimulates oviposition, does not act alone to mediate discrimination among potential host plants, and combination with other water-soluble compounds evoked more potent ovipositional response (Renwick and Radke, 1983). A recent study with a swallowtail butterfly, Papilio xuthus, has also dealt with an analogous topic where vicenin-2 (flavone glycoside), which was inactive alone, proved active when admixed with another water-soluble fraction (Ohsugi et al., 1985). The stimulatory action of naringin and hesperidinon egg deposition by the females appears to prevail only to a limited extent and to be subsidiary, because they neither exhibited any appreciable activity when assayed alone nor played an essential role in oviposition as evidenced by the fact that fraction 5, devoid of the two compounds, did suffice singly to elicit ovipositional response. Accordingly, such flavanone glycosides are not principal determinants of oviposition; nevertheless, either compound should be regarded as factors conducive to the synergy. Although it is difficult to put an explicit interpretation on the enhanced response by ovipositing females to flavanones caused by pretreatment with fraction 5, a possible reason is that this phenomenon was due to "memory effect" brought about by a trace of residual active substances adsorbed on the tarsal chemoreceptors of forelegs. In fact, repetitive washing of foretarsi with water resulted in much reduced response to flavanones. Naringin and hesperidin are not specific to Citrus plants (although not being widespread in plants); however, most Citrus plants have been reported to be excellent producers of these compounds, which are contained not only in fruits but also in leaves (Hattori et al., 1952; Nakabayashi, 1961; Nishiura et al., 1969; Namba et al., 1985). In this respect, it is very likely that Papilio protenor, one of Citrus feeders, has evolutionarily come to rely not on ubiquitous flavonoids such as mtin (flavonol) but on certain specified flavanones in discriminating more precisely its host plants out of a diversity of unacceptable plants. Some Citrus plants are also known to yield several flavones as minor flavonoid constituents. P. protenor, however, made little or no response to such flavone glycosides as rhoifolin and diosmin, the former being frequently encountered in Citrus plants (Hattori et al., 1952; Nakabayashi, 1961; Nishiura et al., 1969). Therefore, flavones in general appear essentially inactive to P. protenor, whereas a sort of flavone glycoside, vicenin-2, has been reported to be active to a related species, P. xuthus (Ohsugi et al., 1985). Feeny et al. (1983) have shown that for P. polyxenes, a umbellifer-feeding swallowtail, naringin and hesperidin were ineffective altogether, while chlorogenic acid and apigenin-7-glucoside (flavone) were slightly active. Such disparity may partly reflect interspecific difference in host preference.
FLAVANONE GLYCOSIDES
2009
In P. p r o t e n o r , all o f t h e c o m p o n e n t s p r e s e n t in a c t i v e f r a c t i o n s s e e m n o t indispensable for inducing oviposition; however, the whole complex of constituents should form the chemical basis for host recognition and preference. T o p o s s e s s a c o m p r e h e n s i v e r e s p o n s e s p e c t r u m to o r a g a i n s t a v a r i e t y o f c o m p o u n d s , n o t r e l y i n g o n a n y s i n g l e s t i m u l a n t , m a y s e r v e to e n s u r e c o r r e c t a n d strict d i s c r i m i n a t i o n o f h o s t p l a n t s b y m o t h e r b u t t e r f l i e s .
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RENWICK, J.A.A., and RADKE,C.D. 1983. Chemical recognition of host plants for oviposition by the cabbage butterfly, Pieris rapae (Lepidoptera: Pieddae). Environ. Entomol. 12:446-450. RODMAN, J.E., and CHEW, F.S. 1980. Phytochemical correlates of herbivory in a community of native and naturalized Cmciferae. Biochem. Syst. Ecol. 8:43-50. ROTHSCHILD, M., and SCHOONHOVEN,L.M. 1977. Assessment of egg load by Pieris brassicae. Nature 266:352-355. SAXENA,K.N., and GOYAL,S. 1978. Host-plant relations of the citrus butterfly Papilio demoleus L.: Orientational and ovipositional responses. Entomol. Exp. Appl. 24:1-10. SHAPIRO, A.M. 1981. Egg-mimics of Streptanthus (Cruciferae) deter oviposition by Pieris sisymbrii. Oecologia 48:142 143. STANTON, M.L. 1979. The role of chemotactile stimuli in the oviposition preferences of Colias butterflies. Oecologia 39:79-91. STANTON,M.L. 1982. Searching in a patchy environment: Food plant selection by Colias p. eriphyle butterflies. Ecology 63: 839-853. VAIDYA,V.G. 1969. investigations of the role of visual stimuli in egg-laying and resting behavior of Papilio demoleus L. (Papilionidae, Lepidoptera). Anita. Behav. 17:350-355. WILLIAMS,K.S., and GILBERT,L.E. 1981. Insects as selective agents on plant vegetative morphology: Egg mimicry reduces egg laying by butterflies. Science 212:467-469.
