Pergamon

PII: S0305-1978(97)00057-4

Biochemical Systematics and Ecology, Vol. 25, No. 6, pp. 527-536,1997 O 1997 Elsevier Science Ltd

All rights reserved. Printed in Great Britain 0305-1978/97 $17.00+0.00

Uptake of Flavonoids from Vicia villosa (Fabaceae) by the Lycaenid Butterfly, Polyommatus icarus (Lepidoptera: Lycaenidae)

FRANK BURGHARDT,* KONRAD FIEDLERI§ and PETER PROKSCHt *Lehrstuhl Verhaltensphysiologie und Soziobiologie, Theodor-Boveri-Biozentrum der Universit&t,

Am Hubland, D-97074 W0rzburg, Germany; l"Lehrstuhl TierOkologie I, Universit~t Bayreuth, D-95440 Bayreuth, Germany;

tLehrstuhl Pharmazeutische Biologie, Julius-von-Sachs-lnstitut f~r Biowissenschaften, Mittlerer Dallenbergweg 64, D-97082 W0rzburg, Germany

Key Word Index--Vicia villosa; Polyommatus icarus; Fabaceae; Lycaenidae; flavonoids; plant-insect interac- tions; wing pigments.

Abstract- -Caterpi l lars of Polyornmatus icarus were reared on inflorescences of Vicia villosa, a plant species acceptable for larval development, but not used as food source in nature. Vicia villosa flowers contained four flavonoids at a concentration of 18.34 mgg -1 dry weight. Three compounds were identified by MS and NMR as rnyricetin-3-O-rhamnoside (8.68%), quemetin-3-O-rhamnoside (56.30%), and kaempferol-3-O-rham- noside (32.18%). Larvae incorporated and metabolized only part of the flavonoids of their hostplant, whi le a larger proportion was excreted. Besides trace amounts of metabolites, adult butterflies contained mainly quer- cetin-3-O-rhamnoside and kaempferol-3-O-rhamnoside at a total concentration of 1 .22-1.38mgg -1. The main f lavonoid in the butterflies was kaempferol-3-O-rhamnoside (55%). Females stored more flavonoids than males, and the f lavonoid content was correlated with body mass. Butterflies raised on V. villosa as larval food differed qualitatively (number and chemical nature of flavonoids) and quantitatively from conspecifics reared on other hostplants. It is proposed that quantitative variation of flavonoids, which are incorporated into the wing patterns, could serve as a means of mate recognition and selection. © 1997 Elsevier Science Ltd

In t roduct ion Flavonoids are ubiquitous phenolic compounds in higher plants with a prominent structural diversity (Harborne, 1991). Therefore, almost all herbivores will encounter these secondary metabolites when feeding. Surprisingly few studies have focused on the fate of flavonoids after ingestion by herbivorous insects (but see Wilson, 1985, 1986, 1987; Hopkins and Ahmad, 1991; Wiesen et aL, 1994). Certain phytophagous insects not only metabolize, but store such plant-derived pigments. This phenomenon is relatively widespread in the Lepidoptera, in particular in butterfly families like the Papi- lionidae, Nymphalidae, and Lycaenidae, where flavonoids are accumulated from the plant diet and stored in the wings or other parts of the body (Ford, 1941 ; Wilson, 1 985, 1986, 1987; Wiesen et al., 1994). In contrast to other secondary plant metabolites, which are accumulated by herbivores for defence (e.g. Bowers, 1993) or pheromone synthesis (e.g. Boppr6, 1990), the biological function of sequestered flavonoids has not been identified so far. Since flavonoids absorb UV light, it has been suggested that these pigments may protect the insects against harmful radiation, or that they may contribute to the wing patterns which they use as visual communication signals (Bernard and Remington, 1991; Meyer-Rochow, 1991 ).

§Corresponding author (Fax: 0921-55-2784; E-maih konrad.fiedler@uni-bayreuth.de).

(Received 25 July 1996; accepted 14 May 1997)

527

528 F. BURGHARDT ETAL.

Insects are unable to synthesize flavonoids or their precursors de novo (Kayser, 1985). Lycaenid butterflies reared on flavonoid-free artificial diets, for example, contain no traces of these phenolics (Wiesen et al., 1994; Burghardt and Fiedler, unpubl.data). Hence, flavonoid uptake and metabolism of insects should strongly depend on the specific flavonoid patterns of their hostplants. Only few studies, however, have so far identified and compared flavonoid patterns of hostplants with those of insects reared on exactly that same diet material (Hopkins and Ahmad, 1991 ; Wiesen et al., 1 994). In an earlier study on a couple of lycaenid butterflies, for example, Wilson (1987) compared flavonoid patterns of wild-caught butterflies (with an unknown feeding history) with flavonoid analyses of putative hostplants (as recorded in the literature). She observed that from all the flavonoids present in the hostplants only flavonols were sequestered by these butterflies. Furthermore, she found evidence for substantial metabolization of fla- vonoids prior to sequestration. Starting from Wilson (1987), we set out to investigate the fate of plant-derived flavonoids in herbivores in more detail, using quantitative HPLC as the main analytical method. A problem of earlier studies was that some of the putative "'hostplant records" were erroneous, whereas certain butterflies are so poly- phagous that without knowledge of the exact feeding history of the analyzed individuals interpretations remain problematic. To overcome such difficulties, we analyzed laboratory-bred insects in parallel with their hostplants.

We have chosen the butterfly family Lycaenidae to further investigate the metabolism of plant-derived flavonoids and their later fate and biological function in the insects for three reasons. (1) It is well established that these insects sequester and store flavonoids (Wilson, 1987; Wiesen et al., 1994). (2) The hostplant relationships of these butterflies are well known, but interactions with secondary plant metabolites remain poorly understood (Fiedler, 1995, 1 996). (3) Earlier studies have revealed that lycaenid but- terflies accumulate most of the flavonoids as part of their wing patterns, that this accu- mulation involves specific uptake and biotransformation of certain compounds, and that the resulting flavonoid profiles differ between the sexes (Wiesen et al., 1 994; Geuder et al., 1997). Therefore, butterflies in the family Lycaenidae are promising models for understanding both the physiological mechanisms and ecological consequences of sequestration of flavonoid pigments in insects.

In the present study, we set out to investigate how a flavonoid-sequestering lycaenid species, the common blue butterfly Polyommatus icarus (Rottemburg, 1775) (Lycae- nidae: Polyommatini), reacts to the flavonoid pattern encountered in a plant species which is acceptable as larval food in the laboratory, but not normally used in the wild. While the fate of flavonoids from natural hostplants is relatively well known in this but- terfly species (Wiesen et al., 1994), it has so far never been studied whether these insects are able to metabolize the flavonoid pattern of a plant to which they are not specifically adapted. As experimental food plant we chose the fodder vetch Vicia villosa Roth (Fabaceae: Vicieae). Polyommatus icarus larvae are relatively polyphagous on inflorescences or young foliage of a wide array of legume species in the tribes Genisteae, Coronilleae, Loteae, Galegeae, and Trifolieae, but the tribe Vicieae is conspicuously absent from the list of natural hostplants of this butterfly (Martin Cano, 1984). Specifi- cally, we asked the following questions: (1) Is Vicia villosa suitable to support the development of P. icarus from the egg to the adult stage? (2) Which compounds of the food plant's flavonoid pattern are present in the insects? (3) Is there evidence for selective accumulation, excretion or biotransformation of certain flavonoids? (4) Do the

UPTAKE OF FLAVONOIDS FROM VICIA VILLOSA 529

sexes differ in their flavonoid accumulation, and how is the flavonoid content of indivi- duals related to body size?

Materials and Methods Insects. Po/yommatus icarus is a widespread Palearctic lycaenid butterfly of open grassland. Larvae feed on a variety of mostly herbaceous Fabaceae species, but are specialized on inflorescences and young tender foliage (Burghardt and Fiedler, 1996). Female butterflies of P, icarus caught in the vicinity of WLirzburg (northern Bavaria) were allowed to oviposit on hostplant flowers (Medicago sa~va) in glass cages kept in a greenhouse. Eggs were collected and transferred into a climatic chamber (15:9 h L:D cycle, 22.5°C constant), in which the entire development took place. After hatching, larvae were first kept in small groups in closed transparent plastic vials (125 ml) lined with moist filter paper. During the final instar, caterpillars were reared singly. Fresh food in excess was provided daily in new clean rearing vials until the larvae pupated. Throughout their entire development, caterpillars were solely fed inflorescences of Vicia vi/Iosa. Pupae were maintained under the same climatic conditions until adult eclosion (for detailed information about the rearing procedure see Schurian (1989) and Burghardt and Fiedler (1996)). After the butterflies had hatched from the pupae, stretched out their wings, and excreted their meconium, they were stored at -20°C and freeze-dried for further work. Samples of larval frass and some fourth instar larvae were also frozen for later analysis. These larvae were kept without food for c. 2-3 h before they were frozen to ensure that their alimentary canal was empty.

Plant material Vicia vi/Iosa is widely distributed in southern and central Europe in open, dry places. To control for seasonal or geographical variability, all plant material used to rear the larvae as well as for subsequent chemical analyses was collected from the same natural plant population at the campus of WOrzburg University in June and July 1994. Inflorescences destined for flavonoid extraction were frozen immediately after col- lection and later freeze-dried.

Chemical analysis of insects, frass, and plant material Freeze-dried butterflies were sexed, weighed, and the length of forewings was measured. Then, each butterfly, caterpillar, or pupa was individually ground in a mortar with methanol. A known calibrated solution of kaempferol (Roth, F.R.G.) was added as internal stan- dard for subsequent quantitative HPLC analysis. To recover the entire fiavonoid content, this mixture was extracted three times over 24h each in 10ml methanol under stirring. Aliquots of the extract (20p, I) were injected into a HPLC system (Gynkotek, F.R.G.) and separated at a flow rate of 1 ml min-1 using an Eurospher column (125mm long and 4.0mm diameter, packed with Nucleosil 100 C 18; 5p, m; Knauer, F.R.G.). Elution was carried out with a leveled gradient consisting of solvent A (H3PO4, aqueous ortho-phosphoric acid; pH 2.0) and solvent B (100% methanol). The gradient started with 90% of solvent A plus 10% of solvent B and ended up with 100% solvent B. Peaks were quantified relative to the internal kaempferol standard. The flavo- holds in the butterflies were analysed by comparative HPLC using retention times and UV spectra of known compounds as reference. Flavonoids were detected by UV at 254nm. Extractions of larvae were basically conducted in the same way separately for each individual, but without sexing. For analyses of larval frass, ali- quots of dried feces were weighed and then processed as above.

Freeze-dried inflorescences of Vicia vi/Iosa (7.0g dry weight) were ground and extracted three times for 24 h with MeOH/H20 (50:50) for preparative isolation of flavonoids. The extract was concentrated and partitioned between MeOH/H20 (50:50) and petrolether, H20 and ethyl acetate. The fractions were inspected by TLC. Fractions which contained flavonoids were applied to a column (Sephadex) with MeOH/CH2CI2 (50:50) as eluent to separate and purify the flavonoids. The three major flavonoid compounds were injected in HPLC to assess purification. The structural identity of the three isolated compounds was proven by MS and NMR spectroscopic methods and by comparison of published data.

Data were evaluated using standard statistical procedures (Student's t-test, Mann-Whitney's U-test, or KruskaI-Wallis test). To test for correlations between flavonoid content and body size, we calculated Spearman's rank correlation coefficient. Throughout the paper, quantitative data are given as arithmetic means + 1 S E. All concentrations are calculated on dry weight basis and given as mg g-1 dry weight (abbre- viated as mgg-1).

Results Suitability of V. villosa as food plant Although caterpillars of P. icarus have not yet been found to feed on V. villosa in nature, they readily accepted this plant and developed without any signs of adverse effects. Prepupae (not separated according to sex) reached a mean fresh weight of 86.96-1- 1.81 mg (n = 25). Fresh weights of adult males (n = 26; 36.88 -t- 1.36 mg) and females (n = 19; 36.98 + 1.76 mg) could not be distinguished from weights reached by butterflies which were reared on natural hostplants under the same conditions (inflor-

530 F. BURGHARDT ETAL.

TABLE 1, SIZE, WEIGHT, AND FLAVONOID CONTENT OF Polyommatus icarus BUTTERFLIES WHOSE LARVAE HAD BEEN RAISED ON Vicia villosa INFLORESCENCES (MEANS ± 1 STANDARD ERROR). Right column presents results of statistical comparisons (one- tailed tests) between the sexes

Female (n = 10) Male (n = 16) Statistics

Forewing length (mm) Dry weight (mg) Fresh weight (mg) Total amount of flavonoids (lig per individual) Concentration of all flavonoids (mg g-1 dry weight) Quercetin-3-O-rhamnoside (#g per individual) Concentration of quercetin-3-O-rhamnoside (rag g- 1 dry weight) Kaempferol-3-O-rhamnoside (~g per individual) Concentration of kaempferol-3-O-rhamnoside (mg g-1 dry weight) Unidentified flavonoid metabolites (lig per individual) Concentration of unidentified flavonoids (mgg -1 dry weight)

15.10 :L 0.17 15,49 ±0.25 t=1.113, p>O.2 13.44±0.50 11.74+0.58 t= 2.037, p= 0.053 31.90±0.92 32.18±1.37 t= 0.149, p> 0.8 18.21 ±1.52 13.27±1.28 t= 2.444, p= 0.022

1.38±0.14 1.12±0.02 t= 1.820, p= 0,081 6.77+0.60 5.00±0.56 Z= 2,266, p<0.03 0.51 ±0.05 0.41 ±0.03 Z= 1.687, p> 0.09

10.42±1.07 7.64 ± 0.78 Z=2.214, p<0.03 0.79±0.10 0.65±0.05 Z= 1.370, p>0.17 1.02±0.13 0.65±0.08 Z= 2.214, p<0.03 0.08+0.01 0.06 ± 0.01 Z= 1.581,p>0.11

escences of Melilotus officinalis: males (n=19): 36.64±0.96mg, females (n=16): 35.87±1.02mg; Medicago sativa: males (n=22): 37.21 +0.95mg, females (n=26): 34.64 ± 1.1 9 mg; KruskaI-Wallis-test: H2df = 0.329; p > 0.8). Since we only extracted a fraction of the reared butterflies for their flavonoid content, the weights given in Table 1 and those reported above slightly differ from one another. There were only minimal, and non-significant, differences between the sexes with regard to forewing length and adult fresh weight (see Fiedler and H011dobler, 1992; Fiedler and Saam, 1994), but female dry weights were significantly higher than those of males (Table 1 ). Weight data of field- caught P. icarus butterflies are unavailable in the literature, but with respect to forewing length the specimens reared on V. villosa are typical for Central European natural popu- lations (13-16mm: Bink (1992)).

Flavonoid patterns of food plant and insect samples In total, inflorescences of V. villosa contained flavonoids at a concentration of

18.34±0.12mgg -1 (Fig. 1). The three major components (Fig. 2A), collectively accounting for >97% of the total flavonoid content of V. villosa, were identified from their spectral data (NMR, MS) as: myricetin-3-O-rhamnoside (1.59mgg -1, 8.68%) (1), quercetin-3-O-rhamnoside (10.32mgg -1, 56.30%) (2), and kaempferol-3-O- rhamnoside (5.90mgg -1, 32.18%) (3). A fourth minor flavonoid component was present in such low concentrations (0.52 mg g - l , 2.84%) that we did not isolate suffi- cient amounts for spectroscopic identification. Judging from its retention time and UV spectrum, however, this component was tentatively identified as rutin (quercetin-3-O- glucoside-O-rhamnoside). Compound (4) in the chromatograms (Fig. 2) denotes the internal kaempferol standard added to the extracts for quantification.

Six fully grown fourth instar caterpillars were extracted for their flavonoid content (Fig. 2B). Besides the three major compounds of the food plant's flavonoid spectrum (myricetin-3-O-rhamnoside (1): 0.2%, 0.004 mg g - l ; quercetin-3-O-rhamnoside (2): 27.1%, 0.53 mg g - l ; kaempferol-3-O-rhamnoside (3): 52.2%, 1.0 mg g - l ) , one other flavonoid (5) was observed which we were unable to identify because of the small amounts available for extraction (20.5%, 0.40 mg g - l ) . The total concentration of fla- vonoids in the caterpillars amounted to 1.955 ± 0.35 mg g - 1, which is only one tenth of the flavonoid concentration in the food plant.

UPTAKE OF FLAVONOIDS FROM VICIA VILLOSA

]2

531

56.30 %

lO

8

"~ 32.18 % 6

2 8.68 %

v / /Z / / / /A 2.84 %

0 ~ ~ ~ ~ querceUn-3-O-rhemnoslde kaem )ferol-3-O-rhamnoalde myricetJn-3-O-n'mmnoeide unknown flavonold

Flavonoid component

FIG. 1. FLAVONOID PATTERN OF Vicie villosa INFLORESCENCES USED AS LABORATORY FOOD PLANT FOR LARVAE OF THE LYCAENID BUTTER FLY, Polyornmatus icarus. Given are means plus one standard error (n = 7 plant samples),

A sample of caterpillar frass combined from a number of individuals (Fig. 2C) was almost as rich in flavonoids as the food plant material (15.8 mg g - l ) . In the feces we detected only those flavonoids which were also present in the diet, in relative amounts very similar to the plant material (myricetin-3-O-rhamnoside: 5.1%, 0.83 mg g - l ; quer- cetin-3-O-rhamnoside: 54%, 8.54mgg-1; kaempferol-3-O-rhamnoside: 41.0%, 6.46 mg g - l ) .

Adult butterflies (Table 1) contained flavonoids at total concentrations of only 1.12 mg g - 1 (males) to 1.38 mg g-1 (females), which was only about two thirds of the flavonoid concentration in larvae and an order of magnitude lower than the flavonoid concentration of the larval diet. The flavonoid pattern of adult butterflies was simplified relative to that of the food plant and the caterpillar (Fig. 2). Two major flavonoids could be identified by comparison with the retention times and spectral data of the plant extracts as quercetin-3-O-rhamnoside (2) and kaempferol-3-O-rhamnoside (3). A small amount of myricetin-3-O-rhamnoside was detected only once among 26 extracted adult butterflies. In addition, we found in all analyzed butterflies 2-3 minor compounds, which together accounted for 4.9% (males) to 5.6% (females) of the total individual flavonoid load. Their UV spectra clearly indicated that these were also flavonoids, and by comparison with their retention times, these unidentified compounds appear to be identical to the metabolites found in the larvae.

Quercetin-3-O-rhamnoside was the main component of the flavonoid pattern in the diet, but in the adult butterflies kaempferol-3-O-rhamnoside was dominant (roughly 57% in both sexes; Table 1). The average share of females on this component was 10.421~g per ind., and for males 7.641~g per ind. Quercetin-3-O-rhamnoside (2) occurred in the butterflies at mean amounts of 6.771ag per ind. (females) and 5.00 pg

532

en

o

o

o

8 o o r

o

J

o o

o

o

o o ~ o

F BURGHARDTETAL.

O o o o o o o

_ZE -,- .~

~_.m

o - -

,o,g

o ---~

2~ o ~ 7o,'

~- d d ~

e ~ O ~ .

<

o ~ ~ g

• 7 o o ° ~

O

UPTAKE OF FLAVONOIDS FROM VICIA VILLOSA 533

per ind. (males), respectively. The concentration of quercetin-3-O-rhamnoside in the butterflies reached only 4.5% of that in the plant, whereas the concentration of kaemp- ferol-3-O-rhamnoside amounted to 12.2% of that observed in the diet. These data suggest that P. icarus larvae reared on V. villosa inflorescences preferentially sequestered the kaempferol derivative over its quercetin analogue or, less likely, metabolized quer- cetin to kaempferol.

Flavonoid content in relation to butterfly sex and size Female P. icarus butterflies reared on V. villosa contained about 37.2% more flavonoids

than males (18.21 I~g vs. 13.271~g, Table 1). This sex difference occurred with the kaempferol- and quercetin-rhamnoside as well as with the unidentified trace metabo- lites. The sex difference in total flavonoid concentration per mg dry weight, however, was only marginally significant (Table 1 ). Nevertheless, since the sex difference in fla- vonoid content was consistently larger (37.2%) than the difference in dry weight (14.48%), these data indicate that P. icarus females sequester flavonoids from V. villosa more strongly than males.

The flavonoid content of butterflies strongly varied with their weight. The total fla- vonoid load per individual butterfly significantly correlated with dry weight (rs = 0.6027, p<O.001, n = 26, both sexes pooled), whereas flavonoid concentrations (either total concentrations or those of particular components) did not correlate with dry weight.

Discussion Our study revealed four results: (1) At least in the laboratory Vicia villosa is a suitable food resource for the butterfly Polyornmatus icarus, which performs on this plant no worse than on natural hosts. (2) The insects incorporate only part of the flavonoids ingested, while most of these phenolics are excreted or metabolized. (3) There is evi- dence for biotransformation of at least some flavonoids. (4) Female butterflies have a higher flavonoid content than males.

Polyommatus icarus larvae use a broad range of legumes as natural hostplants (Martin Cano, 1984) and feeding experiments have already been conducted with other Vicia species (Geiger, 1987). Judging from their weight and size the butterflies bred on V. villosa in the laboratory were equal to specimens raised on natural hostplants or sampled from wild populations. It remains unknown which cues P. icarus females use for host- plant selection and why they apparently reject Vicia species. Our observations indicate that the larvae experience no difficulty in dealing with the flavonoid pattern of V. villosa.

In an earlier study with two other natural hostplant genera Wiesen et al. (1994) have shown that P. icarus butterflies store only a fraction of the flavonoid compounds ingested with larval food. Such also occurred when the insects were raised on the unnatural food plant V. villosa. The flavonoid concentration in larvae amounted to roughly one tenth of the flavonoid concentration in the diet and further decreased towards the adult stage. Certain compounds, like myricetin-3-O-rhamnoside and rutin in our experiments, were completely excreted. Furthermore, the kaempferol-rhamnoside was more effectively stored than its quercetin analogue. This is in accordance with findings of Wilson (1987) and Wiesen et al. (1994) that kaempferol glycosides appear to be the predominant flavonoids found in P. icarus butterflies, although other flavo- holds have been noted, too (Feltwell and Valadon, 1970).

534 F. BURGHARDT ETAL.

We observed two important differences with respect to the study of Wiesen et al. (1994): P. icarus reared on V. villosa exhibited a different flavonoid pattern, and they sequestered distinctly smaller amounts of these phenolics, than conspecifics reared from stock from the same population, but on the natural hostplants Coronilla varia and Med- icago sativa. After development on V. villosa, the adult butterflies contained two major flavonoid compounds (kaempferol-3-O-rhamnoside, quercetin-3-O-rhamnoside) at roughly similar concentrations, whereas specimens reared on C. varia or M. sativa con- sistently had only one major component (kaempferol-3-O-glucoside) supplemented by trace amounts of other flavonoid metabolites. Hence the flavonoid pattern of butterflies raised on the unnatural food plant was more complex. On the other hand, butterflies grown on the two natural hostplants (C. varia, M. sativa) contained more than twice as much flavonoids than those experimentally raised on V. villosa. This difference cannot be attributed to a lower flavonoid supply from the food plant, because the flavonoid content of V. villosa (1.84% of dry wt) was intermediate between the concentrations observed in M. sativa (0.56% of dry wt) and C. varia (3.00% of dry wt). Further experi- ments will be needed to explain why flavonoid storage was that much lower on the experimental food plant despite its apparent suitability in terms of nutrient supply. Indi- vidual or temporal variation of flavonoid content in the various food plant species might also be important here.

Our third finding, i.e. further evidence for biotransformation of flavonoids, was not unexpected since metabolites not present in the larval food had already been reported from P. icarus larvae and adults by Wiesen et al. (1994). Such metabolites accounted for a rather large fraction (20.5%) of the larval flavonoid content, but persisted only as trace compounds (5% altogether) into the adult stage. No metabolites were recorded in the adults which had not already been present in the larvae, suggesting that most flavonoid biotransformation occurs during the larval stage in P. icarus (see also Wiesen et al., 1994). In the related oligophagous species P. bellargus, in contrast, significant biocon- version of flavonoids occurs during the pupal stage (Geuder et al., 1997).

Polyommatus icarus females consistently contained about 37% larger amounts of fla- vonoids than males. A similar difference (non-significant due to small sample sizes) had already been observed by Wiesen et al. (1994). Since female dry weights were higher than those of males, and because flavonoid content correlated well with dry mass, it could be argued that this difference is largely a consequence of sexual size differences. In accordance with this interpretation, concentrations of flavonoids per dry weight were only marginally higher in females than in males. However, the lead of females over males in flavonoid content (37%) was more than twice as high as the weight difference (14,5%), and sexual size and weight differences are generally small and unstable in P. icarus (Fiedler and H611dobler, 1992; Fiedler and Saam, 1994). It seems therefore pos- sible that female P. icarus usually sequester flavonoids more intensively than males, but this requires further confirmation. In the related P. bellargus, the difference in flavonoid sequestration between females and males is much more pronounced (Geuder et al., 1997).

The flavonoid content of adult P. icarus butterflies shows considerable qualitative as well as quantitative individual variation. This variation largely depends on the larval food plant (Ononis sp.: Feltwell and Valadon (1970); Medicago sativa, Coronilla varia: Wiesen et al. (1994); Vicia villosa: this study). In a relatively polyphagous species like P. icarus, the number and precise chemical identity of flavonoid pigments incorporated into the

UPTAKE OF FLAVONOIDS FROM VICIA VILLOSA 535

w i n g s are p robab ly not s ign i f i can t for these butterf l ies. The quan t i t y of stored p igments, however , m igh t p rov ide v isual in fo rmat ion t h rough the in tens i ty of w i n g patterns in UV l ight. UV pat terns are k n o w n to be impor tan t for species d isc r im ina t ion and mate se lect ion in var ious but ter f l ies (Si lberg l ied, 1984; M e y e r - R o c h o w , 1991; Bernard and Reming ton , 1991) . The lycaen id but ter f ly P, icarus preferably ut i l izes f l avono id - r i ch in f lorescences of var ious legumes as larval food, and f l ower - fed ind iv idua ls at ta in larger w e i g h t s and size than fo l iage- fed s ib l ings (Bu rghard t and Fiedler, 1996) . Therefore, a h igh f l avono id load m igh t serve to ind icate a h igher f i tness of potent ia l mates.

Acknowledgements---We thank B. G0ssregen and A. Kunze for their help in the laboratory. The comments of two anonymous reviewers significantly improved the manuscript. MS analyses were kindly carried out by Dr L. Witte (Braunschweig, Germany), and NMR spectra were kindly provided by Dr V. Wray (Braunschweig, Germany). Financial support by the Deutsche Forschungsgemeinschaft to K. Fiedler and P. Proksch (Fi 547/2- 1 ) and the Fonds der chemischen Industrie to P. Proksch is gratefully acknowledged.

R e f e r e n c e s Bernard, G. D. and Remington, C. L. (1991) Color vision in Lycaena butterflies: spectral tuning of receptor

arrays in relation to behavioral ecology. Proc. NatlAcad. ScL U.S.A. 88, 2738-2787. Bink, F. A. (1992) Ecologische Atlas van de L)agvlinders van Noordwest-Europa, 512 pp. Schuyt & Co.,

Haarlem. Boppr~, M. (1990) Lepidoptera and pyrrolizidine alkaloids. Exemplification of complexity in chemical ecology.

J. Chem. EcoL 16, 165-185. Bowers, M D. (1993) Aposematic caterpillars: life-styles of the warningly colored and unpalatable. In Cater-

pillars--Ecological and Evolutionary Constraints on Foraging, eds N. E. Stamp and T. M. Casey, pp. 331-371. Chapman & Hall, New York.

Burghardt, F. and Fiedler, K. (1996) The influence of diet on growth and secretion behaviour of myrmeco- philous Polyommatus icarus caterpillars (Lepidoptera: Lycaenidae). EcoL Entomol. 21, 1-8.

Feltwell, J. and Valadon, L. R. G. (1970) Plant pigments identified in the common blue butterfly. Nature 225, 969.

Fiedler, K. (1995) Lycaenid butterflies and plants: is myrmecophily associated with particular hostplant pre- ferences? EthoL EcoL Evol. 7, 107-132.

Fiedler, K. (1996) Hostplant relationships of lycaenid butterflies: large-scale patterns, interactions with plant chemistry, and mutualism with ants. Entomol. exp. appL 80, 259-267.

Fiedler, K. and H611dobler, B. (1992) Ants and Polyommatus icarus immatures (Lycaenidae): sex-related developmental benefits and costs of ant-attendance. Oecologia 91,468-473.

Fiedler, K. and Saam, C. (1994) Does ant-attendance influence development in 5 European lycaenid butterfly species? Nota lepid. 17, 5-24.

Ford, E. B. (1941) Studies on the chemistry of pigments in the Lepidoptera with reference to their bearing on systematics. 1. The anthoxanthins. Proc. R. Entomol. Soc. London Set. A 16, 65-90.

Geiger, W. (ed.) (1987) Tagfalter und ihre Lebensrbume. Schweizerischer Bund for Naturschutz, Basel. Geuder, M., Wray, V., Fiedler, K. and Proksch, P. (1997) Sequestration and metabolism of host-plant flavo-

holds by the lycaenid butterfly Polyommatus bellargus. J. Chem. Ecol. (in press). Harborne, J. B. (1991) Flavonoid pigments. In Herbivores." their Interaction with Secondary Plant Metabolites,

eds G. A. Rosenthal and M. R. Berenbaum, 2nd Edn, Vol. 1, pp. 389-429. Academic Press, San Diego. Hopkins, T. L. and Ahmad, S. A. (1991 ) Flavonoid wing pigments in grasshoppers. Experientia 47, 1089-1091. Kayser, H. (1985) Pigments. In Comprehensive Insect Physiology, Biochemistry and Pharmacology vol. 10: Bio-

chemistry, eds G. A. Kerkut and L. I. Gilbert. Pergamon Press, Oxford. Martin Cano, J. (1984) Biologia comparada de Lampides boeticus (L.), Syntarucus pirithous (L.) y Polyom-

matus icarus (Rot.) (Lep. Lycaenidae). Graellsia 40, 163-193. Meyer-Rochow, V. B. (1991 ) Differences in ultraviolet wing patterns in the New Zealand lycaenid butterflies

Lycaena salustius, L. rauparaha and L. feredayi as a likely isolating mechanism. J. R. Soc. N. Zealand 21, 169- 177.

Schurian, K. G. (1989) Revision der Lysandra-Gruppe des Genus Polyommatus Latr. (Lepidoptera: Lycae- nidae). Neue entomol. Nachr. 24, 1-181.

Silberglied, R. E. (1984) Visual communication and sexual selection among butterflies. In The Biology of But- terflies, eds R. I. Vane-Wight and P. R. Ackery, pp. 207-223. Academic Press, London.

Wiesen, B., Krug, E., Fiedler, K., Wray, V. and Proksch, P. (1994) Sequestration of host-plant-derived flavo- noids by lycaenid butterfly Polyommatus icarus, J. Chem. Ecol, 20, 2523-2538.

Wilson, A. (1985) Flavonoid pigments of butterflies in the genus Melanargia. Phytochemistry 24, 1685-1691.

536 F. BURGHARDT ETAL.

Wilson, A. (1986) Flavonoid pigments in swallowtail butterflies. Phytochemistry 25, 1309-1313. Wilson, A. (1987) Flavonoid pigments in chalkhill blue Lysandra coridon (Poda) and other lycaenid butterflies.

J. Chem. Ecol. 13, 473-493.