Journal of Chemical Ecology, l,'ol. 21, No. 2, 1995
SEQUESTRATION OF LICHEN COMPOUNDS BY THREE SPECIES OF TERRESTRIAL SNAILS L
SONJA HESBACHER, 2 BRUNO PETER
BAUR, 3 ANETTE
B A U R 3 and
P R O K S C H 2'*
~Julius-~,on-Sachs-lnstitut far Biowissenschasqen Universitiit W~i~burg Mittlerer Dallenbergweg 64 D-97082 Warzburg, Germany 3Zoologisches lnstitut Universitat Basel Rheinsprung 9 CH-4051 Basel, Switzerland (Received May 24, 1994; accepted November 1, 1994)
Abstract--Three species of lichen-grazing snails, Balea perversa, Chondrina clienta, and Helicigona lapicida, all l'rom the Swedish island of Oland, were found to sequester lichen compounds when feeding on the crustous lichen species Aspicila calcarea, Caloplaca flavovirescens, Lecanora muralis, Physcia adscendens, Tephromela atra, and Xanthoria parietina. The lichen compounds detected in the soft bodies of the snail species analyzed included the anthraquinone parietin, the depside atranorin, as well as a presumable degradation product of the latter. Other lichen compounds such as (+)-usnic acid or et-cotlatolic acid were not found in the soft bodies but were only detected in the feces, suggesting selective uptake of lichen compounds by the snails. In individuals of C. clienta initially fed on the lichen X. parietina, the amount of sequestered parietin decreased over time on a parietin-free diet but was still detectable in the soft bodies after 28 days. In the ovoviviparous land snail, B. perversa, sequestered parietin was transferred from the mother to the eggs in the reproductive tract. Key Words--Balea perversa, Chondrina clienta, Helicigona lapicida, Gastropoda, Pulmonata, crustous lichens, lichen compounds, sequestration.
*To whom correspondence should be addressed. I Dedicated to Prof. Dr. F.-C. Czygan on the occasion of his 60th birthday. 233 0098-0331/95/0200-0233507.50/0 O 1995PlenumPublishingCorporation
234
HESBACHER ET AL. INTRODUCTION
Lichens comprise some 20,000 different taxa and show a worldwide distribution (Crittenden and Porter, 1991). In habitats with harsh environmental conditions (e.g., hot or cold deserts) that tend to suppress the growth of most higher plants, lichens can constitute dominant floral elements and may provide important nutritional resources for herbivores. It was speculated some 100 years ago that the observed unpalatability of lichens to most potential herbivores is largely due to chemical defense relying on the accumulation of copious amounts of lichen compounds (Zukal, 1895; Stahl, 1904), whereas other authors, including for example Zopf (1896), denied a defensive function of lichen compounds. Recent years, however, have witnessed growing experimental evidence in favor of the chemical defense hypothesis (Slansky, 1979; Lawrey, 1986, 1989, 1991). For example, experiments with larvae of the vigorous polyphagous herbivore Spodoptera littoralis (Noctuidae) demonstrated pronounced acute toxicity and feeding deterrency for ( - ) and (+)-usnic acid and for vulpinic acid at concentrations well below those found in lichen thalli (Emmerich et al., 1993). Other lichen compounds such as oxyphysodic acid, fumarprotocetraric acid, or calycin, which had no acute effects on survival, were found to retard larval growth and to cause developmental malformations of adults of S. littoralis originating from larvae that were reared on an artificial diet spiked with the respective lichen compounds at concentrations comparable to those found in lichen thalli (Giez et al., 1994). The recent studies suggest that lichen compounds act as defense agents against generalist herbivores. However, there are numerous examples of specialized herbivores that feed solely on lichens. Important groups of oligophagous invertebrate lichen feeders include oribatid mites (Seyd and Seaward, 1984; Reutimann and Scheidegger, 1987), insects (especially Lepidoptera of the family Arctiidae) (Rambold, 1985), and terrestrial gastropods (Lawrey, 1986; Baur et al., 1992). How do these specialized lichen feeders cope with the toxic or unpalatable secondary compounds of their food source that effectively deter generalist herbivores (Emmerich et al., 1993; Giez et al., 1994)? Several strategies of dealing with potentially harmful compounds present in the food source have been elucidated in interactions of herbivores with higher plants. Specialized herbivores may, for example, overcome the defensive chemistry of their hosts by rapid excretion of harmful metabolites or by metabolic detoxification (Brattsten, 1986). Another strategy of dealing with harmful plant compounds frequently observed in specialized herbivores is sequestration of the respective compounds, as shown, for example, for arctiid moths that sequester pyrrolizidine alkaloids from host plants (Ehmke et al., 1990), for cardenolides that are sequestered by monarch butterflies (Malcolm, 1990), or for flavonoids sequestered by lycaenid butterflies (Wiesen et al., 1994). The sequestered plant compounds are often
SEQUESTRATION OF LICHEN COMPOUNDS
235
used for the chemical defense of the herbivores (Ehmke et al., 1990; Malcolm, 1990). As shown for the first time in the present study, specialized lichen-feeding snails (Balea perversa, Chondrina clienta, and Helicigona lapicida) are also capable of storing lichen compounds derived from their hosts. The ecological role of the sequestered lichen compounds in the snails, however, remains open. In addition to the comparative analysis of the sequestered lichen compounds, we present experimental evidence for the transfer of sequestered parietin from the mother to the offspring in the reproductive tract of the ovoviviparous snail
B. perversa.
METHODS AND MATERIALS
Snail Species. Chondrina clienta (Westerlund) occurs in open limestone areas of central and southeastern Europe and in three isolated areas of Sweden (Kerney and Cameron, 1979). Its cylindroconical shell is dextral and in adults is 5.5-7 mm high (Baur, 1988). Balea perversa (L.) is characteristic of dry places among rocks and old stone walls, occurring occasionally on stems of trees. It is widespread in western Europe, and in Scandinavia it occurs mainly along the coast (Kerney and Cameron, 1979). Its narrowly conical shell is sinistral and in adults is 7-10 mm high. Helicigona lapicida (L.) is common in holes and crevices in rocky ground and in old woodland and hedgerows in western and central Europe. Fully grown snails have a flattened, sharply keeled shell measuring 12-20 mm in diameter. All three species are particularly well adapted to rocky habitats; they are very resistant to drought, with activity confined to periods of high air humidity, and their specialized radulae enable them to graze epi- and endolithic lichens from rock faces (Schmid 1929; Breure and Gittenberger, 1982). In the limestone grassland Great Alvar on the Baltic island of ()land (Sweden), 108 species of calcicotous lichens have been recorded (Fr6berg, 1989). On a limestone pavement in this grassland, 17 species of terrestrial gastropods have been found, four of them being potential lichen feeders (Chondrina clienta, Balea perversa, Clausilia bidentata, Pupilla muscorum) (Baur, 1987). Laboratory experiments demonstrated that individuals of C. clienta grazed on 18 of 27 lichen species offered, whereas B. perversa fed on all eight lichen species offered (Fr6berg et al., 1993). Experiments also showed that these two snail species differed in their lichen preferences and that juveniles of both snail species exhibited differences in growth rate when fed on different lichen diets (Bauer et al., 1994). There is some experimental evidence that both intra- and interspecific competition for lichens occur between these snail species (Baur, 1988, 1990; Baur and Baur, 1990).
236
HESBACHER ET AL.
Lichen Compounds in Wild-Caught Snails. Specimens of the land snails C. clienta, B. perversa, and H. lapicida were collected at seven sites in the limestone grassland Great Alvar on 01and in August 1993: (1) in fissures of a vertical quarry wall, 1.8 m high, 1.5 km SSW of Vickleby church (H. lapicida), (2) a stone wall, 0.9 m high, made of flat pieces of limestone arranged in horizontal layers, 40 m S of site 1 (C. clienta, B. perversa); (3) in cracks of a limestone pavement (50 m × 40 m wide), 10 km E of Mrrbylanga (C. clienta); (4) a stone wall adjacent to site 3, 1 m high, made of flat pieces of limestone (B. perversa); (5) in cracks of a limestone pavement (100 m x 120 m wide), 2.5 km N of Fr6sslunda (C. clienta); (6) on a pile of stones (4 m × 25 m wide, 1 m high), 1.5 km N of Frrsslunda (C. clienta); and (7) a stone wall, 1 m high, separated by a 4-m-wide grass strip from the stone pile of site 6 (B. perversa). To exclude any contamination by ingested lichen tissue, the snails collected were starved by keeping them active for four days on moist paper toweling. This time was found to be sufficient for the snails to empty their digestive tract of ingested lichen material as shown by light-microscopic inspection of dissected snails. HPLC was used to analyze sequestered lichen compounds in groups of four snails each. Feeding Experiment. A controlling feeding experiment was designed to examine whether snails sequester particular lichen compounds. Six species of crustous lichens--Aspicila calcarea (L.) Mudd, Caloplacaflavovirescens (Wulfen) Dalla Torre & Samth., Lecanora muralis (Schreber) Rabenh., Physcia adscendens (Fr.) H. Olivier, Tephromela atra (Hudson) Hafellner, and Xanthoria parietina (L.) Th. Fr.--that occur in the snails natural habitats were collected in the surroundings of the Ecological Research Station of Uppsala University in Skogsby approximately 8 km N of site 1. Specimens of snails were collected at site 1 (14. lapicida) and site 2 ( C. clienta and B. perversa; see above). Snails of all three species were fed a cyanobacteria diet during a conditioning period of six days. During this period, as well as in succeeding experiments, the snails were kept at room temperature (20-22°C) and under natural light conditions. Following a starvation period of eight days (as described above), four individuals of either C. clienta, B. perversa, or two individuals of H. lapicida were placed in a transparent plastic dish (6.5 cm diameter, 2 cm height) lined with paper toweling. A piece of lichen (approximately 3 cm in diameter) was placed on top. This set-up was repeated for each of the six lichen species. Snails of all three species are active only during periods of high air humidity, during which they graze lichens (Schmid, 1929; Neucket, 1981). To stimulate periodic snail activity, the lichens and paper toweling were moistened and the dishes covered with an acetate film; after 12 h, the waterproof cover was exchanged for a paper towel to dry out the dishes. This procedure was carried out repeatedly at 48-hr intervals, resulting in a rhythm of alternating 12 hr activity and 36 hr resting.
SEQUESTRATION OF LICHEN COMPOUNDS
237
After a feeding period of eight days, the snails were starved for four days (as described above), and the feces collected. Lichen samples, feces, and soft bodies of snails were analyzed for lichen compounds using HPLC. HPLC Analysis. Lichens as well as feces were ground, extracted in a known aliquot of acetone, and directly subjected to HPLC analysis. Soft bodies of the snails were usually freeze-dried before extraction with known aliquots of acetone. The HPLC system was from Pharmacia (Sweden) and was equipped with a photodiode army detector (Waters). Samples were injected on a Nova-Pak C~8 column (Waters) (150 × 3.9 mm, 4-/zm pore size) and separated using a linear gradient from 100% A (10% MeOH, 90% H20 adjusted to pH 2 with orthophosphoric acid) to 100% B (MeOH) in 40 min. Identification of lichen compounds was by comparison of retention times and by comparison of the on-linerecorded UV absorption spectra with those of commercially available lichen compounds (Roth) or with lichen compounds isolated and identified previously. Lichen compounds were quantified by the external standard method using commercially available lichen products (Roth). Retention o f Sequestered Parietin. Another laboratory experiment was conducted to examine whether sequestered parietin (1 in Figure 1) is retained over a long period in snails feeding on a parietin-free diet. Specimens of C. clienta were collected from a vertical rock wall close to site 2 on August 8, 1993, and samples of X. parietina were collected in the surroundings of the Ecological Research Station. Twelve groups of snails, each consisting of 14 individuals, were kept on a X. parietina diet for 10 days. The snails were maintained as described in the feeding experiment (see above). After this period, the snails were randomly assigned to two treatments. Half of the snails were continuously kept on a X. parietina diet and the remaining snails were fed a parietin-free diet (free-living cyanobacteria growing on small pieces of limestone). Samples of snails were obtained from both treatments at the beginning of the experiment and after each of four successive periods of eight days. These snails were starved for four days to empty their digestive tract before they were analyzed for sequestered parietin using HPLC. Sequestered Parietin in Newborn Snails. B. perversa is ovoviviparous, i.e., the young emerge from the egg in the reproductive tract of the mother (Baur, 1990). To examine whether sequestered parietin is passed to embryos in the reproductive tract of the mother, we analyzed neonate B. perversa with no feeding experience. Fifty adult B. perversa were collected at site 4 on August 8, 1993. The snails were kept in 10 groups each consisting of five individuals on a X. parietina diet for 10 days. Maintenance of snails was as described in the feeding experiment (see above). Following this conditioning period, the snails were repeatedly kept on moist paper toweling (no food available) for two days followed by a X. parietina diet for two days during a total period of 56 days. Snails born in the parietin-free environment (N = 5t) were collected for
238
HESBACHER ET AL,
HO
O
OH
C H 3 0 ~ C H O
~
OHC
OH
CH3
2
H,-O~c.-/CH3 0I ~ 0..~.~
HoH~coocH3
H_C" "U
CH3
,JLY
\H-'""
3
. O"H -c
L~3
4
CH2CO%Hll
OH
¢~2cocs~lx 5 FIo. 1. Structures of lichen compounds identified: 1, parietin; 2, atranorin; 3, presumed degradation product of 2; 4, usnic acid, (+)-enantiomer; 5, c~-coUatolicacid.
SEQUESTRATION OF LICHEN COMPOUNDS
239
HPLC analysis of sequestered parietin. For each analysis, 8-12 neonate snails were used. RESULTS
Lichen Compounds in Wild-Caught Snails. Table 1 summarizes the sequestered lichen compounds found in specimens of three snail species. HPLC analysis of the snails revealed the presence of the lichen compounds parietin (1, Figure 1), atranorin (2, Figure 1) and of the monomer (3, Figure 1)--the latter presumably originating by hydrolytic cleavage of atranorin--in B. perversa from two localities. Compounds 2 and 3 were also detected in specimens of C. ctienta from two localities. Snails of the species H. lapicida were devoid of lichen compounds. However, specimens of the latter were only available from one natural habitat, whereas B. perversa and C. clienta could be collected at three localities each. Since the lichen compounds or their metabolites were detected in snails that were starved following collection to exclude contamination by ingested lichen tissue, a sequestration of lichen compounds must be assumed at least for B. perversa and C. clienta. Feeding Experiment. Table 2 summarizes the lichen compounds found in six lichen species and in the soft bodies of C. clienta, B. perversa, and H. lapicida that had fed on the respective lichens. The feces of the three snail species usually resembled those of the corresponding lichen species with regard to the profiles of lichen compounds (e.g., Figures 2 and 3). Lichen products were also present in the soft bodies of the snails analyzed even though the total amounts (in micrograms per snail) of individual compounds showed considerable
T A B L E 1. LICHEN C O M P O U N D S IN SOFT BODIES AND FECES OF W I L D - C A U G H T SNAILS
Lichen compounds(numbersas in Figure 1) Snails
Collection sites"
In soft bodies
In feces
B. perversa
4 6 7
1,2,3 1,2 --
1,3 1,3 1
C. clienta
2 3 6
-2 2,3
----
H. lapicida
1
--
--
"For descriptionof collectionssites see Methods and Materials.
B. perversa C. clienta H. lapicida B, perversa C, clienta 1t= tapicida B. perversa C. clienta H, lapicida B, perversa C. clienta H, lapicida B. perversa C. clienta #t. lapicida B. perversa C, clienta 1t, lapicida
Snails
0.13 0.02 0.15
0.02 + 0,06
0.06
Amount (,ug/ind.)
10.98 3.57 0.07
1,7 + 0.03
12.3
Conc (,~g/g) fresh wt
1
0,08
16.40
+
+
+
+
Conc (#g/g) fresh wt
Amount (#g/ind.)
2
19.43 + 0.11
0,23
3.38 92.25 0.18 3,38 + +
+
2.53 18.45 0,11 +
33,80 4.10 +
Conc (~g/g) fresh wt
0.23 +
0.04 0.45 0.37 0,04 + +
+
0.03 0.09 0,22 +
0.40 0.02 +
Amount (#g/ind.)
"Numbers of lichen compounds are as in Figure 1, Compounds 4 and 5 were not detected in soft bodies of snails. ( + ) = trace amount.
X. parietina (1, 2)
T, atra (2, 3, 5)
P. adscendens (2, 3)
L, muralis (2, 4)
C, flavovirescens (1, 2)
A. calcarea (1, 2)
Lichens used for feeding experiments; identified lichen compounds are given in parentheses
Identified lichen compounds in soft bodies of snails
FEEDING ON SIX DIFFERENT SPECIES OF LICHENSa
TABLE 2. DISTRIBUTION AMOUNTS, AND CONCENTRATIONS OF LICHEN COMPOUNDS IN SOFT BODIES OF SNAILS FOLLOWING
> r"
cl
'I,
o
i,o
241
SEQUESTRATION OF LICHEN COMPOUNDS
0 i
10 i
i
i
,
i
i
,
,
,
i
20 . . . .
i
. . . .
[
30 . . . .
i
.
~
,
i
i
i
i
,
i
I
FIG. 2. Comparative HPLC analysis of soft bodies (C) and feces (B) of specimens of Chondrina clienta after feeding on thalli of Physcia adscendens (A). Specimens of C. clienta were treated as described in the feeding experiment. Numbers of compounds are as in Figure t.
intra- and interspecific variation for the snail species studied. Relatively large amounts of lichen products were detected in specimens of C. clienta that fed on P. adscendens (Table 2). The major lichen compound detected in the snails was the monomer 3 (0,45 izg/snail), whereas its presumable precursor atranorin (2) (0.08/~g/snail) was dominant in both the thallus and feces (Figure 2), suggesting preferential sequestration of the monomer 3 by the snails vs. the depside atranorin (2). Similar results that suggest a preferential sequestration of the monomer (3) compared to its presumable precursor atranorin (2) were found in
242
HESBACHER ET AL.
A
C
0 i
,
,
, ~
,la
10 JJ . . . .
20 i
. . . .
30 i
. . . .
,
. . . .
,~l
,
,r
FIG. 3. Comparative HPLC analysis of soft bodies (C) and feces (B) of specimens of Balea perversa after feeding on thalli of Lecanora muralis (A). Specimens of B. perversa were treated as described in the feeding experiment. Numbers of compounds are as in Figure 1. individuals of B. perversa and H. lapicida that fed on P. adscendens. The monomer (3) was the only detectable lichen compound in snails of both species (0.37 ~tg/snail for H. lapicida and 0.04 ttg/snail for B. perversa), whereas the feces contained both atranorin (2) and the monomer (3) (data not shown). The anthraquinone parietin (1) was another lichen compound detected in specimens of B. perversa (0.02-0.13 #g/snail), C. clienta (trace-0.06 /~g/snail), and H. lapicida (0.06-0.15/~g/snail) that fed on thalli of C. flavovirescens or X. parietina, which both contain parietin (Table 2). Other lichen compounds, however,
243
SEQUESTRATION OF LICHEN COMPOUNDS
such as c~-collatolic acid (5) (present in T. atra) or (+)-usnic acid (4) (present in L. muralis) were only found in the feces but were not detected in any soft body of the snails analyzed, as exemplified for B. perversa in Figure 3. These findings suggest a preferential sequestration of parietin (1), atranorin (2), and monomer (3) by the snails when compared to ct-collatolic acid (5) or (+)-usnic acid (4). Retention of Sequestered Parietin (1). Following an initial feeding period of 10 days on X. parietina, individuals of C. clienta were kept either on a parietin-free diet (cyanobacteria) or on a X. parietina diet for another 28 days. Subsamples of six snails were removed from both groups at intervals of eight days and analyzed for sequestered parietin (1) (Figure 1). The total amount of sequestered parietin (approximately 0.10-0.14/zg/snail) remained almost constant in the group of snails kept on a X. parietina diet during the whole experiment, whereas in the group that fed on cyanobacteria the amount of sequestered parietin decreased with time. However, even after being kept on a parietin-free diet for 28 days, the snails still contained traces of the sequestered anthraquinone (0.01 #g/snail compared to 0. I0/zg/snail at the beginning of the experiment) (Figure 4). Sequestered Parietin in Newborn Snails. Sequestered parietin (1) was passed on to newborn B. perversa that had no feeding experience on lichens. Adult B. perversa that were kept on a X. parietina diet for 10-66 days gave birth to young that contained parietin at amounts ranging from 2.8 to 3.9 ng/snail (equivalent to concentrations of 3.8-5.3/~g/g fresh wt).
z
0.10-
v
z I-I.,U
0.05-
(b
O-
0
I
I
1
I
I
2
3
4
WEEKS
FIG. 4. Time-dependent decrease in the amount of sequestered parietin (1) in individuals of Balea perversa when fed a parietin-free diet (A). Snails of group B were constantly fed on Xanthoria parietina.
244
HESBACHER ET AL. DISCUSSION
The present study provides, to our knowledge for the first time, evidence for a sequestration of lichen compounds by lichen-feeding terrestrial gastropods. The lichen compounds parietin (1), atranorin (2), or the presumable degradation product (3) were found (at least in trace amounts) in most of the soft bodies analyzed, whereas both (+)-usnic acid (4) as well as c~-collatolic acid (5) were apparently discriminated by the snails (Table 2 and Figure 3). Light-microscopic analysis of dissected snails indicated that a starvation period of four days (as used in the feeding experiments) was sufficient to empty the digestive tract of lichen material. The preferential uptake of parietin (1) or atranorin (2) vs. (+)-usnic acid (4), for example, seems not to be due to differences in lipophilicity, which may influence diffusion across membranes. When subjected to partitioning between H20 and CH2C12 (the latter representing a lipophilic compartment) almost 100% of compounds 1-5 are recovered from the lipophilic phase (as measured by HPLC; data not shown), indicating that there are no significant differences between the compounds with regard to lipophilicity. Hence, there must be other, so far unknown reasons for the observed differences in sequestration of the various lichen compounds. The discrimination of (+)-usnic acid (4) by all of the snails studied (Table 2, Figure 3) is of special interest with respect to the established toxicity of this compound towards invertebrate herbivores. For example, when injected into last-instar larvae of the vigorous insect herbivore Spodoptera littoralis (Noctuidae), (+)-usnic acid (4) caused pronounced larval mortality (LDso 70 mg/kg body wt), whereas both parietin (1) and atranorin (2) proved to be nontoxic to the larvae (Emmerich et al., 1993; Giez et al., 1994); c~-Collatolic acid (5) has not been studied for toxicity towards invertebrates so far. It is possible that the toxicity of (+)-usnic acid (4) is not restricted to insects such as S. littoralis but extends to other invertebrate grazers including snails. Effective discrimination of usnic acid compared to the demonstrated sequestration of both parietin and atranorin may possibly enable snails to employ even potentially harmful lichens such as L. muralis as a food source without a risk of intoxication. It is so far unknown whether the sequestered lichen substances are of ecological importance to the snails. However, it has been shown that anthraquinone derivatives such as chrysazin and chrysophanol, which are structurally closely related to parietin (1), are deterrent towards ants of the species Myrmica sabuleti (Hilker et al., 1992). Chrysazin and chrysophanol are used by various chrysomelid beetles for their chemical defense and have also been suggested to protect the eggs of the beeries (Hilker et al., 1992). Marine gastropods of the molluscan subclass Opisthobranchia (the so-called nudibranchs) are notorious for the sequestration of secondary products from dietary sources such as sponges, which in turn are utilized by the nudibranchs for their own chemical defense against
SEQUESTRATION OF LICHEN COMPOUNDS
245
p r e d a t o r s s u c h as c a r n i v o r o u s fishes ( H a y a n d F e n i c a l , 1988; R o d g e r s and Paul 1991; F a u l k n e r , 1992; P r o k s c h , 1994). It is t h e r e f o r e p o s s i b l e that s t o r a g e o f l i c h e n c o m p o u n d s m a y result in c h e m i c a l p r o t e c t i o n o f terrestrial snails f r o m p r e d a t o r s . In this c o n t e x t it is o f interest to note that s e v e r a l s p e c i e s o f birds o n the island o f O l a n d f e e d o n e m p t y shells o f the snails u n d e r study but s e e m to a v o i d live s p e c i m e n s , s u g g e s t i n g d e t e r r e n c y that m i g h t b e c a u s e d by s e q u e s t e r e d lichen c o m p o u n d s ( B a u r et al., u n p u b l i s h e d ) . F u r t h e r s t u d i e s o n the e c o l o g i c a l role o f s e q u e s t e r e d lichen c o m p o u n d s in terrestrial snails are u n d e r w a y . Acknowledgments--We thank the staff at the Ecological Research Station of Uppsala University on ()land for their hospitality and Dr. L, Frfberg (Lund, Sweden) as well as Dr. B. Bfidel and Prof. O.L. Lange (both Wiirzburg, Germany) for help in determining the lichen species. Financial support was received from the Deutsche Forschungsgemeinschaft as a project of the "'Sonderforschungsbereich 251 der Universit~it Wfir'zberg (to P.P.) and from the Swiss National Science Foundation (grant 31-33511.92 to B.B.), and the Treubel Foundation of the Freiwitlige Akademische Gesellschaft, University of Basel (grant to B.B.).
REFERENCES BAUR, B. 1987. Richness of land snail species under isolated stones in a karst area on Oland, Sweden. Basteria 51 : 129-133. BAUR, B. 1988. Microgeographical variation in shell size of the land snail Chondrina clienta. Biol. J. Linn. Soc. 35:247-259. BAUR, B. t990. Intra- and interspecific influences on age at first reproduction and fecundity in the land snail Belea perversa. Oikos 57:333-337. BAUR, B., and BAUR, A. 1990. Experimental evidence for intra- and interspecific competition in two species of rock-dwelling land snails. J. Anita. Ecol. 59:301-315. BAUR, A., BAUR, B., and FROBERG, L. 1992. The effect of lichen diet on growth rate in the rockdwelling land snails Chondrina clienta (Westerlund) and Balea perversa (Linnaeus). J. Moll. Stud. 58:345-347. BAUR, A., BAUR, B., and FROBERG, L. 1994. Herbivory on calcicolous lichens: different food preferences and growth rates in two co-existing land snails. Oecologia 98:313-319. BRA~rrSTEN, L.B. 1986. Fate of ingested plant aUelochemicals in herbivorous insects, pp. 211-255, L.B. Brattsten, S. Ahmad, in Molecular Aspects of Insect-Plant Associations. Plenum Press, New York. BREURE, A.S.H., and GIq"rENBERGER,E. 1982. The rock-scraping radula, a striking case of convergence (Mollusca). Neth. J. Zool. 32:307-312. CRr~ENDEN, P.D., and PORTER, N. 1991. Lichen forming fungi: potential sources of novel metabolites. Tib. Tech. 9:409-414. EHMKE, A., W[TTE, L., BILLER, A., and HARTMANN, T. 1990. Sequestration, N-oxidation and transformation of plant pyrrolizidine alkaloids by the Arctiid moth Tyria jacobaeae L. Z. Naturforsch. 45c: 1185-1192. EMMZRICH, R., GIEZ, I., LANCE, O.L., and PROKSCH, P. 1993, Toxicity and antifeedant activity of lichen compounds against the polyphagous herbivorous insect Spodoptera littoralis. Phytochernistry 33:1389-1394. FAULKNER, D.J. 1992. Chemical defenses of marine molluscs, pp. 119-163, (V.J. Paul, ed.). in Ecological Roles of Marine Natural Products. Comstock, Ithaca.
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HESBACHER ET AL.
FROBERG, L. 1989. The calcicolous lichens on the Great Alvar of 0land, Sweden. PhD thesis. University of Lund. FROBERG, L., BAUR, Ao, and BAr0R, B. 1993. Differential herbivore damage to calcicolous lichens by snails. Lichenologist 25:83-95. Gmz, I., LANGE, O.L., and PROKSCH, P. 1994. Growth retarding activity of lichen substances against the polyphagous herbivorous insect Spodoptera littoralis. Biochem. Syst. Ecol. 22:113120. HAY, M.E., and FENtCAL, W. 1988. Marine plant-herbivore interactions: The ecology of chemical defense. Ann. Rev, Ecol. Syst. 19:111-145. HILKER, M., ESCHBACH, U., and DETTNER, K. 1992. Occurrence of anthraquinones in eggs and larvae of several Galerucinae (Coleoptera: Chrysomelidae). Naturwissenschafien 79:271-274. KERNEY, M,P., and CAMERON, R.A.D. 1979. A Field Guide to the Land Snails of Britain and North-west Europe. Collins, London. LAWREY, J.D. 1986. Biological roles of lichen substances, B~.ologist 89:111-122. LAWREY, LD, 1989. Lichen secondary compounds: Evidence for a correspondence between antiherbivore and antimicrobial function. Bryologist 92:326-328. LAWREY, J,D. 1991, Biotic interactions in lichen community development: A review. Lichenologist 23:205-214. MALCOLM, S.B. 1990. Chemical defense in chewing and sucking insect herbivores: Plant derived cardenolides in the monarch butterfly and oleander aphid. Chemoecology l:12-21. NUECKEL, W. 1981. Zu Aktivit~itsregelung und Wasserhaushalt von Chondrina avenacea (Bruguiere 1792), einer Felsen bewohnenden Landlungenschnecke. PhD thesis. University of Basel. PROKSCH, P. 1994. Defensive roles for secondary metabolites from marine sponges and spongefeeding nudibranchs. Toxicon 32:639-655. RAMBOLD, G. 1985. Fiitterungsexperimente mit den an Flechten lebenden Raupen von Setina aurita Esp, Nachrichtenblatt bayer. Entomology 34:82-90~ REUTIMANN,P., and SCHE1DEGGER,C. 1987. Importance of lichen secondary products in food choice of two oribatid mites (Acari) in an Alpine meadow ecosystem. J, Chem. Ecol. 13:363-370. RODGERS, S.D., and PAUL, V.J. 1991. Chemical defenses of three Glossodoris nudibranchs and their dietary Hyrtios sponges. Mar. EcoL Progr. Ser. 77:221-232. SCHMID, G. 1929. Endolithische Kalkflechten und Schneckenfrass, BioL ZentralbL 49:28-35. SEYD, E.L., and SEAWARD, M.R.D. 1984. The association of oribatid mites with lichens. ZooL J. Linn. Soc. 80:369-420. SLANSKY, F. 1979. Effect of the lichen chemicals atranorin and vulpinic acid upon feeding and growth of larvae of the yellow-striped armyworm Spodoptera ornithogalli. Environ. Entomot. 8:865-868. STAHL, E. 1904. Die Schutzmittel der Flechten gegen Tieffrass, pp. 357-375, in Festschrift zum 70. Geburtstag yon Ernst Haeckel. G. Fischer Verlag, Jena. WIESEN, B., KRUG, E., FIEDLER, K., WRAY, V,, and PROKSCH, P. 1994. Sequestration of hostplant derived flavonoids by the Lycaenid butterfly Polyommatus icarus. J. Chem, Ecol. 20:25232538. ZOPF, W, 1896. Flechtenstoffe. G, Fischer Verlag, Jena, ZUKAL. H. 1895. Morphotogische und biologische Untersuchungen fiber die Flechten. Sber. K. BOhm. Ges. Wiss. Math. -Nat. KI. 104:1303-1395.
SEQUESTRATION OF LICHEN COMPOUNDS BY THREE SPECIES OF TERRESTRIAL SNAILS L
SONJA HESBACHER, 2 BRUNO PETER
BAUR, 3 ANETTE
B A U R 3 and
P R O K S C H 2'*
~Julius-~,on-Sachs-lnstitut far Biowissenschasqen Universitiit W~i~burg Mittlerer Dallenbergweg 64 D-97082 Warzburg, Germany 3Zoologisches lnstitut Universitat Basel Rheinsprung 9 CH-4051 Basel, Switzerland (Received May 24, 1994; accepted November 1, 1994)
Abstract--Three species of lichen-grazing snails, Balea perversa, Chondrina clienta, and Helicigona lapicida, all l'rom the Swedish island of Oland, were found to sequester lichen compounds when feeding on the crustous lichen species Aspicila calcarea, Caloplaca flavovirescens, Lecanora muralis, Physcia adscendens, Tephromela atra, and Xanthoria parietina. The lichen compounds detected in the soft bodies of the snail species analyzed included the anthraquinone parietin, the depside atranorin, as well as a presumable degradation product of the latter. Other lichen compounds such as (+)-usnic acid or et-cotlatolic acid were not found in the soft bodies but were only detected in the feces, suggesting selective uptake of lichen compounds by the snails. In individuals of C. clienta initially fed on the lichen X. parietina, the amount of sequestered parietin decreased over time on a parietin-free diet but was still detectable in the soft bodies after 28 days. In the ovoviviparous land snail, B. perversa, sequestered parietin was transferred from the mother to the eggs in the reproductive tract. Key Words--Balea perversa, Chondrina clienta, Helicigona lapicida, Gastropoda, Pulmonata, crustous lichens, lichen compounds, sequestration.
*To whom correspondence should be addressed. I Dedicated to Prof. Dr. F.-C. Czygan on the occasion of his 60th birthday. 233 0098-0331/95/0200-0233507.50/0 O 1995PlenumPublishingCorporation
234
HESBACHER ET AL. INTRODUCTION
Lichens comprise some 20,000 different taxa and show a worldwide distribution (Crittenden and Porter, 1991). In habitats with harsh environmental conditions (e.g., hot or cold deserts) that tend to suppress the growth of most higher plants, lichens can constitute dominant floral elements and may provide important nutritional resources for herbivores. It was speculated some 100 years ago that the observed unpalatability of lichens to most potential herbivores is largely due to chemical defense relying on the accumulation of copious amounts of lichen compounds (Zukal, 1895; Stahl, 1904), whereas other authors, including for example Zopf (1896), denied a defensive function of lichen compounds. Recent years, however, have witnessed growing experimental evidence in favor of the chemical defense hypothesis (Slansky, 1979; Lawrey, 1986, 1989, 1991). For example, experiments with larvae of the vigorous polyphagous herbivore Spodoptera littoralis (Noctuidae) demonstrated pronounced acute toxicity and feeding deterrency for ( - ) and (+)-usnic acid and for vulpinic acid at concentrations well below those found in lichen thalli (Emmerich et al., 1993). Other lichen compounds such as oxyphysodic acid, fumarprotocetraric acid, or calycin, which had no acute effects on survival, were found to retard larval growth and to cause developmental malformations of adults of S. littoralis originating from larvae that were reared on an artificial diet spiked with the respective lichen compounds at concentrations comparable to those found in lichen thalli (Giez et al., 1994). The recent studies suggest that lichen compounds act as defense agents against generalist herbivores. However, there are numerous examples of specialized herbivores that feed solely on lichens. Important groups of oligophagous invertebrate lichen feeders include oribatid mites (Seyd and Seaward, 1984; Reutimann and Scheidegger, 1987), insects (especially Lepidoptera of the family Arctiidae) (Rambold, 1985), and terrestrial gastropods (Lawrey, 1986; Baur et al., 1992). How do these specialized lichen feeders cope with the toxic or unpalatable secondary compounds of their food source that effectively deter generalist herbivores (Emmerich et al., 1993; Giez et al., 1994)? Several strategies of dealing with potentially harmful compounds present in the food source have been elucidated in interactions of herbivores with higher plants. Specialized herbivores may, for example, overcome the defensive chemistry of their hosts by rapid excretion of harmful metabolites or by metabolic detoxification (Brattsten, 1986). Another strategy of dealing with harmful plant compounds frequently observed in specialized herbivores is sequestration of the respective compounds, as shown, for example, for arctiid moths that sequester pyrrolizidine alkaloids from host plants (Ehmke et al., 1990), for cardenolides that are sequestered by monarch butterflies (Malcolm, 1990), or for flavonoids sequestered by lycaenid butterflies (Wiesen et al., 1994). The sequestered plant compounds are often
SEQUESTRATION OF LICHEN COMPOUNDS
235
used for the chemical defense of the herbivores (Ehmke et al., 1990; Malcolm, 1990). As shown for the first time in the present study, specialized lichen-feeding snails (Balea perversa, Chondrina clienta, and Helicigona lapicida) are also capable of storing lichen compounds derived from their hosts. The ecological role of the sequestered lichen compounds in the snails, however, remains open. In addition to the comparative analysis of the sequestered lichen compounds, we present experimental evidence for the transfer of sequestered parietin from the mother to the offspring in the reproductive tract of the ovoviviparous snail
B. perversa.
METHODS AND MATERIALS
Snail Species. Chondrina clienta (Westerlund) occurs in open limestone areas of central and southeastern Europe and in three isolated areas of Sweden (Kerney and Cameron, 1979). Its cylindroconical shell is dextral and in adults is 5.5-7 mm high (Baur, 1988). Balea perversa (L.) is characteristic of dry places among rocks and old stone walls, occurring occasionally on stems of trees. It is widespread in western Europe, and in Scandinavia it occurs mainly along the coast (Kerney and Cameron, 1979). Its narrowly conical shell is sinistral and in adults is 7-10 mm high. Helicigona lapicida (L.) is common in holes and crevices in rocky ground and in old woodland and hedgerows in western and central Europe. Fully grown snails have a flattened, sharply keeled shell measuring 12-20 mm in diameter. All three species are particularly well adapted to rocky habitats; they are very resistant to drought, with activity confined to periods of high air humidity, and their specialized radulae enable them to graze epi- and endolithic lichens from rock faces (Schmid 1929; Breure and Gittenberger, 1982). In the limestone grassland Great Alvar on the Baltic island of ()land (Sweden), 108 species of calcicotous lichens have been recorded (Fr6berg, 1989). On a limestone pavement in this grassland, 17 species of terrestrial gastropods have been found, four of them being potential lichen feeders (Chondrina clienta, Balea perversa, Clausilia bidentata, Pupilla muscorum) (Baur, 1987). Laboratory experiments demonstrated that individuals of C. clienta grazed on 18 of 27 lichen species offered, whereas B. perversa fed on all eight lichen species offered (Fr6berg et al., 1993). Experiments also showed that these two snail species differed in their lichen preferences and that juveniles of both snail species exhibited differences in growth rate when fed on different lichen diets (Bauer et al., 1994). There is some experimental evidence that both intra- and interspecific competition for lichens occur between these snail species (Baur, 1988, 1990; Baur and Baur, 1990).
236
HESBACHER ET AL.
Lichen Compounds in Wild-Caught Snails. Specimens of the land snails C. clienta, B. perversa, and H. lapicida were collected at seven sites in the limestone grassland Great Alvar on 01and in August 1993: (1) in fissures of a vertical quarry wall, 1.8 m high, 1.5 km SSW of Vickleby church (H. lapicida), (2) a stone wall, 0.9 m high, made of flat pieces of limestone arranged in horizontal layers, 40 m S of site 1 (C. clienta, B. perversa); (3) in cracks of a limestone pavement (50 m × 40 m wide), 10 km E of Mrrbylanga (C. clienta); (4) a stone wall adjacent to site 3, 1 m high, made of flat pieces of limestone (B. perversa); (5) in cracks of a limestone pavement (100 m x 120 m wide), 2.5 km N of Fr6sslunda (C. clienta); (6) on a pile of stones (4 m × 25 m wide, 1 m high), 1.5 km N of Frrsslunda (C. clienta); and (7) a stone wall, 1 m high, separated by a 4-m-wide grass strip from the stone pile of site 6 (B. perversa). To exclude any contamination by ingested lichen tissue, the snails collected were starved by keeping them active for four days on moist paper toweling. This time was found to be sufficient for the snails to empty their digestive tract of ingested lichen material as shown by light-microscopic inspection of dissected snails. HPLC was used to analyze sequestered lichen compounds in groups of four snails each. Feeding Experiment. A controlling feeding experiment was designed to examine whether snails sequester particular lichen compounds. Six species of crustous lichens--Aspicila calcarea (L.) Mudd, Caloplacaflavovirescens (Wulfen) Dalla Torre & Samth., Lecanora muralis (Schreber) Rabenh., Physcia adscendens (Fr.) H. Olivier, Tephromela atra (Hudson) Hafellner, and Xanthoria parietina (L.) Th. Fr.--that occur in the snails natural habitats were collected in the surroundings of the Ecological Research Station of Uppsala University in Skogsby approximately 8 km N of site 1. Specimens of snails were collected at site 1 (14. lapicida) and site 2 ( C. clienta and B. perversa; see above). Snails of all three species were fed a cyanobacteria diet during a conditioning period of six days. During this period, as well as in succeeding experiments, the snails were kept at room temperature (20-22°C) and under natural light conditions. Following a starvation period of eight days (as described above), four individuals of either C. clienta, B. perversa, or two individuals of H. lapicida were placed in a transparent plastic dish (6.5 cm diameter, 2 cm height) lined with paper toweling. A piece of lichen (approximately 3 cm in diameter) was placed on top. This set-up was repeated for each of the six lichen species. Snails of all three species are active only during periods of high air humidity, during which they graze lichens (Schmid, 1929; Neucket, 1981). To stimulate periodic snail activity, the lichens and paper toweling were moistened and the dishes covered with an acetate film; after 12 h, the waterproof cover was exchanged for a paper towel to dry out the dishes. This procedure was carried out repeatedly at 48-hr intervals, resulting in a rhythm of alternating 12 hr activity and 36 hr resting.
SEQUESTRATION OF LICHEN COMPOUNDS
237
After a feeding period of eight days, the snails were starved for four days (as described above), and the feces collected. Lichen samples, feces, and soft bodies of snails were analyzed for lichen compounds using HPLC. HPLC Analysis. Lichens as well as feces were ground, extracted in a known aliquot of acetone, and directly subjected to HPLC analysis. Soft bodies of the snails were usually freeze-dried before extraction with known aliquots of acetone. The HPLC system was from Pharmacia (Sweden) and was equipped with a photodiode army detector (Waters). Samples were injected on a Nova-Pak C~8 column (Waters) (150 × 3.9 mm, 4-/zm pore size) and separated using a linear gradient from 100% A (10% MeOH, 90% H20 adjusted to pH 2 with orthophosphoric acid) to 100% B (MeOH) in 40 min. Identification of lichen compounds was by comparison of retention times and by comparison of the on-linerecorded UV absorption spectra with those of commercially available lichen compounds (Roth) or with lichen compounds isolated and identified previously. Lichen compounds were quantified by the external standard method using commercially available lichen products (Roth). Retention o f Sequestered Parietin. Another laboratory experiment was conducted to examine whether sequestered parietin (1 in Figure 1) is retained over a long period in snails feeding on a parietin-free diet. Specimens of C. clienta were collected from a vertical rock wall close to site 2 on August 8, 1993, and samples of X. parietina were collected in the surroundings of the Ecological Research Station. Twelve groups of snails, each consisting of 14 individuals, were kept on a X. parietina diet for 10 days. The snails were maintained as described in the feeding experiment (see above). After this period, the snails were randomly assigned to two treatments. Half of the snails were continuously kept on a X. parietina diet and the remaining snails were fed a parietin-free diet (free-living cyanobacteria growing on small pieces of limestone). Samples of snails were obtained from both treatments at the beginning of the experiment and after each of four successive periods of eight days. These snails were starved for four days to empty their digestive tract before they were analyzed for sequestered parietin using HPLC. Sequestered Parietin in Newborn Snails. B. perversa is ovoviviparous, i.e., the young emerge from the egg in the reproductive tract of the mother (Baur, 1990). To examine whether sequestered parietin is passed to embryos in the reproductive tract of the mother, we analyzed neonate B. perversa with no feeding experience. Fifty adult B. perversa were collected at site 4 on August 8, 1993. The snails were kept in 10 groups each consisting of five individuals on a X. parietina diet for 10 days. Maintenance of snails was as described in the feeding experiment (see above). Following this conditioning period, the snails were repeatedly kept on moist paper toweling (no food available) for two days followed by a X. parietina diet for two days during a total period of 56 days. Snails born in the parietin-free environment (N = 5t) were collected for
238
HESBACHER ET AL,
HO
O
OH
C H 3 0 ~ C H O
~
OHC
OH
CH3
2
H,-O~c.-/CH3 0I ~ 0..~.~
HoH~coocH3
H_C" "U
CH3
,JLY
\H-'""
3
. O"H -c
L~3
4
CH2CO%Hll
OH
¢~2cocs~lx 5 FIo. 1. Structures of lichen compounds identified: 1, parietin; 2, atranorin; 3, presumed degradation product of 2; 4, usnic acid, (+)-enantiomer; 5, c~-coUatolicacid.
SEQUESTRATION OF LICHEN COMPOUNDS
239
HPLC analysis of sequestered parietin. For each analysis, 8-12 neonate snails were used. RESULTS
Lichen Compounds in Wild-Caught Snails. Table 1 summarizes the sequestered lichen compounds found in specimens of three snail species. HPLC analysis of the snails revealed the presence of the lichen compounds parietin (1, Figure 1), atranorin (2, Figure 1) and of the monomer (3, Figure 1)--the latter presumably originating by hydrolytic cleavage of atranorin--in B. perversa from two localities. Compounds 2 and 3 were also detected in specimens of C. ctienta from two localities. Snails of the species H. lapicida were devoid of lichen compounds. However, specimens of the latter were only available from one natural habitat, whereas B. perversa and C. clienta could be collected at three localities each. Since the lichen compounds or their metabolites were detected in snails that were starved following collection to exclude contamination by ingested lichen tissue, a sequestration of lichen compounds must be assumed at least for B. perversa and C. clienta. Feeding Experiment. Table 2 summarizes the lichen compounds found in six lichen species and in the soft bodies of C. clienta, B. perversa, and H. lapicida that had fed on the respective lichens. The feces of the three snail species usually resembled those of the corresponding lichen species with regard to the profiles of lichen compounds (e.g., Figures 2 and 3). Lichen products were also present in the soft bodies of the snails analyzed even though the total amounts (in micrograms per snail) of individual compounds showed considerable
T A B L E 1. LICHEN C O M P O U N D S IN SOFT BODIES AND FECES OF W I L D - C A U G H T SNAILS
Lichen compounds(numbersas in Figure 1) Snails
Collection sites"
In soft bodies
In feces
B. perversa
4 6 7
1,2,3 1,2 --
1,3 1,3 1
C. clienta
2 3 6
-2 2,3
----
H. lapicida
1
--
--
"For descriptionof collectionssites see Methods and Materials.
B. perversa C. clienta H. lapicida B, perversa C, clienta 1t= tapicida B. perversa C. clienta H, lapicida B, perversa C. clienta H, lapicida B. perversa C. clienta #t. lapicida B. perversa C, clienta 1t, lapicida
Snails
0.13 0.02 0.15
0.02 + 0,06
0.06
Amount (,ug/ind.)
10.98 3.57 0.07
1,7 + 0.03
12.3
Conc (,~g/g) fresh wt
1
0,08
16.40
+
+
+
+
Conc (#g/g) fresh wt
Amount (#g/ind.)
2
19.43 + 0.11
0,23
3.38 92.25 0.18 3,38 + +
+
2.53 18.45 0,11 +
33,80 4.10 +
Conc (~g/g) fresh wt
0.23 +
0.04 0.45 0.37 0,04 + +
+
0.03 0.09 0,22 +
0.40 0.02 +
Amount (#g/ind.)
"Numbers of lichen compounds are as in Figure 1, Compounds 4 and 5 were not detected in soft bodies of snails. ( + ) = trace amount.
X. parietina (1, 2)
T, atra (2, 3, 5)
P. adscendens (2, 3)
L, muralis (2, 4)
C, flavovirescens (1, 2)
A. calcarea (1, 2)
Lichens used for feeding experiments; identified lichen compounds are given in parentheses
Identified lichen compounds in soft bodies of snails
FEEDING ON SIX DIFFERENT SPECIES OF LICHENSa
TABLE 2. DISTRIBUTION AMOUNTS, AND CONCENTRATIONS OF LICHEN COMPOUNDS IN SOFT BODIES OF SNAILS FOLLOWING
> r"
cl
'I,
o
i,o
241
SEQUESTRATION OF LICHEN COMPOUNDS
0 i
10 i
i
i
,
i
i
,
,
,
i
20 . . . .
i
. . . .
[
30 . . . .
i
.
~
,
i
i
i
i
,
i
I
FIG. 2. Comparative HPLC analysis of soft bodies (C) and feces (B) of specimens of Chondrina clienta after feeding on thalli of Physcia adscendens (A). Specimens of C. clienta were treated as described in the feeding experiment. Numbers of compounds are as in Figure t.
intra- and interspecific variation for the snail species studied. Relatively large amounts of lichen products were detected in specimens of C. clienta that fed on P. adscendens (Table 2). The major lichen compound detected in the snails was the monomer 3 (0,45 izg/snail), whereas its presumable precursor atranorin (2) (0.08/~g/snail) was dominant in both the thallus and feces (Figure 2), suggesting preferential sequestration of the monomer 3 by the snails vs. the depside atranorin (2). Similar results that suggest a preferential sequestration of the monomer (3) compared to its presumable precursor atranorin (2) were found in
242
HESBACHER ET AL.
A
C
0 i
,
,
, ~
,la
10 JJ . . . .
20 i
. . . .
30 i
. . . .
,
. . . .
,~l
,
,r
FIG. 3. Comparative HPLC analysis of soft bodies (C) and feces (B) of specimens of Balea perversa after feeding on thalli of Lecanora muralis (A). Specimens of B. perversa were treated as described in the feeding experiment. Numbers of compounds are as in Figure 1. individuals of B. perversa and H. lapicida that fed on P. adscendens. The monomer (3) was the only detectable lichen compound in snails of both species (0.37 ~tg/snail for H. lapicida and 0.04 ttg/snail for B. perversa), whereas the feces contained both atranorin (2) and the monomer (3) (data not shown). The anthraquinone parietin (1) was another lichen compound detected in specimens of B. perversa (0.02-0.13 #g/snail), C. clienta (trace-0.06 /~g/snail), and H. lapicida (0.06-0.15/~g/snail) that fed on thalli of C. flavovirescens or X. parietina, which both contain parietin (Table 2). Other lichen compounds, however,
243
SEQUESTRATION OF LICHEN COMPOUNDS
such as c~-collatolic acid (5) (present in T. atra) or (+)-usnic acid (4) (present in L. muralis) were only found in the feces but were not detected in any soft body of the snails analyzed, as exemplified for B. perversa in Figure 3. These findings suggest a preferential sequestration of parietin (1), atranorin (2), and monomer (3) by the snails when compared to ct-collatolic acid (5) or (+)-usnic acid (4). Retention of Sequestered Parietin (1). Following an initial feeding period of 10 days on X. parietina, individuals of C. clienta were kept either on a parietin-free diet (cyanobacteria) or on a X. parietina diet for another 28 days. Subsamples of six snails were removed from both groups at intervals of eight days and analyzed for sequestered parietin (1) (Figure 1). The total amount of sequestered parietin (approximately 0.10-0.14/zg/snail) remained almost constant in the group of snails kept on a X. parietina diet during the whole experiment, whereas in the group that fed on cyanobacteria the amount of sequestered parietin decreased with time. However, even after being kept on a parietin-free diet for 28 days, the snails still contained traces of the sequestered anthraquinone (0.01 #g/snail compared to 0. I0/zg/snail at the beginning of the experiment) (Figure 4). Sequestered Parietin in Newborn Snails. Sequestered parietin (1) was passed on to newborn B. perversa that had no feeding experience on lichens. Adult B. perversa that were kept on a X. parietina diet for 10-66 days gave birth to young that contained parietin at amounts ranging from 2.8 to 3.9 ng/snail (equivalent to concentrations of 3.8-5.3/~g/g fresh wt).
z
0.10-
v
z I-I.,U
0.05-
(b
O-
0
I
I
1
I
I
2
3
4
WEEKS
FIG. 4. Time-dependent decrease in the amount of sequestered parietin (1) in individuals of Balea perversa when fed a parietin-free diet (A). Snails of group B were constantly fed on Xanthoria parietina.
244
HESBACHER ET AL. DISCUSSION
The present study provides, to our knowledge for the first time, evidence for a sequestration of lichen compounds by lichen-feeding terrestrial gastropods. The lichen compounds parietin (1), atranorin (2), or the presumable degradation product (3) were found (at least in trace amounts) in most of the soft bodies analyzed, whereas both (+)-usnic acid (4) as well as c~-collatolic acid (5) were apparently discriminated by the snails (Table 2 and Figure 3). Light-microscopic analysis of dissected snails indicated that a starvation period of four days (as used in the feeding experiments) was sufficient to empty the digestive tract of lichen material. The preferential uptake of parietin (1) or atranorin (2) vs. (+)-usnic acid (4), for example, seems not to be due to differences in lipophilicity, which may influence diffusion across membranes. When subjected to partitioning between H20 and CH2C12 (the latter representing a lipophilic compartment) almost 100% of compounds 1-5 are recovered from the lipophilic phase (as measured by HPLC; data not shown), indicating that there are no significant differences between the compounds with regard to lipophilicity. Hence, there must be other, so far unknown reasons for the observed differences in sequestration of the various lichen compounds. The discrimination of (+)-usnic acid (4) by all of the snails studied (Table 2, Figure 3) is of special interest with respect to the established toxicity of this compound towards invertebrate herbivores. For example, when injected into last-instar larvae of the vigorous insect herbivore Spodoptera littoralis (Noctuidae), (+)-usnic acid (4) caused pronounced larval mortality (LDso 70 mg/kg body wt), whereas both parietin (1) and atranorin (2) proved to be nontoxic to the larvae (Emmerich et al., 1993; Giez et al., 1994); c~-Collatolic acid (5) has not been studied for toxicity towards invertebrates so far. It is possible that the toxicity of (+)-usnic acid (4) is not restricted to insects such as S. littoralis but extends to other invertebrate grazers including snails. Effective discrimination of usnic acid compared to the demonstrated sequestration of both parietin and atranorin may possibly enable snails to employ even potentially harmful lichens such as L. muralis as a food source without a risk of intoxication. It is so far unknown whether the sequestered lichen substances are of ecological importance to the snails. However, it has been shown that anthraquinone derivatives such as chrysazin and chrysophanol, which are structurally closely related to parietin (1), are deterrent towards ants of the species Myrmica sabuleti (Hilker et al., 1992). Chrysazin and chrysophanol are used by various chrysomelid beetles for their chemical defense and have also been suggested to protect the eggs of the beeries (Hilker et al., 1992). Marine gastropods of the molluscan subclass Opisthobranchia (the so-called nudibranchs) are notorious for the sequestration of secondary products from dietary sources such as sponges, which in turn are utilized by the nudibranchs for their own chemical defense against
SEQUESTRATION OF LICHEN COMPOUNDS
245
p r e d a t o r s s u c h as c a r n i v o r o u s fishes ( H a y a n d F e n i c a l , 1988; R o d g e r s and Paul 1991; F a u l k n e r , 1992; P r o k s c h , 1994). It is t h e r e f o r e p o s s i b l e that s t o r a g e o f l i c h e n c o m p o u n d s m a y result in c h e m i c a l p r o t e c t i o n o f terrestrial snails f r o m p r e d a t o r s . In this c o n t e x t it is o f interest to note that s e v e r a l s p e c i e s o f birds o n the island o f O l a n d f e e d o n e m p t y shells o f the snails u n d e r study but s e e m to a v o i d live s p e c i m e n s , s u g g e s t i n g d e t e r r e n c y that m i g h t b e c a u s e d by s e q u e s t e r e d lichen c o m p o u n d s ( B a u r et al., u n p u b l i s h e d ) . F u r t h e r s t u d i e s o n the e c o l o g i c a l role o f s e q u e s t e r e d lichen c o m p o u n d s in terrestrial snails are u n d e r w a y . Acknowledgments--We thank the staff at the Ecological Research Station of Uppsala University on ()land for their hospitality and Dr. L, Frfberg (Lund, Sweden) as well as Dr. B. Bfidel and Prof. O.L. Lange (both Wiirzburg, Germany) for help in determining the lichen species. Financial support was received from the Deutsche Forschungsgemeinschaft as a project of the "'Sonderforschungsbereich 251 der Universit~it Wfir'zberg (to P.P.) and from the Swiss National Science Foundation (grant 31-33511.92 to B.B.), and the Treubel Foundation of the Freiwitlige Akademische Gesellschaft, University of Basel (grant to B.B.).
REFERENCES BAUR, B. 1987. Richness of land snail species under isolated stones in a karst area on Oland, Sweden. Basteria 51 : 129-133. BAUR, B. 1988. Microgeographical variation in shell size of the land snail Chondrina clienta. Biol. J. Linn. Soc. 35:247-259. BAUR, B. t990. Intra- and interspecific influences on age at first reproduction and fecundity in the land snail Belea perversa. Oikos 57:333-337. BAUR, B., and BAUR, A. 1990. Experimental evidence for intra- and interspecific competition in two species of rock-dwelling land snails. J. Anita. Ecol. 59:301-315. BAUR, A., BAUR, B., and FROBERG, L. 1992. The effect of lichen diet on growth rate in the rockdwelling land snails Chondrina clienta (Westerlund) and Balea perversa (Linnaeus). J. Moll. Stud. 58:345-347. BAUR, A., BAUR, B., and FROBERG, L. 1994. Herbivory on calcicolous lichens: different food preferences and growth rates in two co-existing land snails. Oecologia 98:313-319. BRA~rrSTEN, L.B. 1986. Fate of ingested plant aUelochemicals in herbivorous insects, pp. 211-255, L.B. Brattsten, S. Ahmad, in Molecular Aspects of Insect-Plant Associations. Plenum Press, New York. BREURE, A.S.H., and GIq"rENBERGER,E. 1982. The rock-scraping radula, a striking case of convergence (Mollusca). Neth. J. Zool. 32:307-312. CRr~ENDEN, P.D., and PORTER, N. 1991. Lichen forming fungi: potential sources of novel metabolites. Tib. Tech. 9:409-414. EHMKE, A., W[TTE, L., BILLER, A., and HARTMANN, T. 1990. Sequestration, N-oxidation and transformation of plant pyrrolizidine alkaloids by the Arctiid moth Tyria jacobaeae L. Z. Naturforsch. 45c: 1185-1192. EMMZRICH, R., GIEZ, I., LANCE, O.L., and PROKSCH, P. 1993, Toxicity and antifeedant activity of lichen compounds against the polyphagous herbivorous insect Spodoptera littoralis. Phytochernistry 33:1389-1394. FAULKNER, D.J. 1992. Chemical defenses of marine molluscs, pp. 119-163, (V.J. Paul, ed.). in Ecological Roles of Marine Natural Products. Comstock, Ithaca.
246
HESBACHER ET AL.
FROBERG, L. 1989. The calcicolous lichens on the Great Alvar of 0land, Sweden. PhD thesis. University of Lund. FROBERG, L., BAUR, Ao, and BAr0R, B. 1993. Differential herbivore damage to calcicolous lichens by snails. Lichenologist 25:83-95. Gmz, I., LANGE, O.L., and PROKSCH, P. 1994. Growth retarding activity of lichen substances against the polyphagous herbivorous insect Spodoptera littoralis. Biochem. Syst. Ecol. 22:113120. HAY, M.E., and FENtCAL, W. 1988. Marine plant-herbivore interactions: The ecology of chemical defense. Ann. Rev, Ecol. Syst. 19:111-145. HILKER, M., ESCHBACH, U., and DETTNER, K. 1992. Occurrence of anthraquinones in eggs and larvae of several Galerucinae (Coleoptera: Chrysomelidae). Naturwissenschafien 79:271-274. KERNEY, M,P., and CAMERON, R.A.D. 1979. A Field Guide to the Land Snails of Britain and North-west Europe. Collins, London. LAWREY, J.D. 1986. Biological roles of lichen substances, B~.ologist 89:111-122. LAWREY, LD, 1989. Lichen secondary compounds: Evidence for a correspondence between antiherbivore and antimicrobial function. Bryologist 92:326-328. LAWREY, J,D. 1991, Biotic interactions in lichen community development: A review. Lichenologist 23:205-214. MALCOLM, S.B. 1990. Chemical defense in chewing and sucking insect herbivores: Plant derived cardenolides in the monarch butterfly and oleander aphid. Chemoecology l:12-21. NUECKEL, W. 1981. Zu Aktivit~itsregelung und Wasserhaushalt von Chondrina avenacea (Bruguiere 1792), einer Felsen bewohnenden Landlungenschnecke. PhD thesis. University of Basel. PROKSCH, P. 1994. Defensive roles for secondary metabolites from marine sponges and spongefeeding nudibranchs. Toxicon 32:639-655. RAMBOLD, G. 1985. Fiitterungsexperimente mit den an Flechten lebenden Raupen von Setina aurita Esp, Nachrichtenblatt bayer. Entomology 34:82-90~ REUTIMANN,P., and SCHE1DEGGER,C. 1987. Importance of lichen secondary products in food choice of two oribatid mites (Acari) in an Alpine meadow ecosystem. J, Chem. Ecol. 13:363-370. RODGERS, S.D., and PAUL, V.J. 1991. Chemical defenses of three Glossodoris nudibranchs and their dietary Hyrtios sponges. Mar. EcoL Progr. Ser. 77:221-232. SCHMID, G. 1929. Endolithische Kalkflechten und Schneckenfrass, BioL ZentralbL 49:28-35. SEYD, E.L., and SEAWARD, M.R.D. 1984. The association of oribatid mites with lichens. ZooL J. Linn. Soc. 80:369-420. SLANSKY, F. 1979. Effect of the lichen chemicals atranorin and vulpinic acid upon feeding and growth of larvae of the yellow-striped armyworm Spodoptera ornithogalli. Environ. Entomot. 8:865-868. STAHL, E. 1904. Die Schutzmittel der Flechten gegen Tieffrass, pp. 357-375, in Festschrift zum 70. Geburtstag yon Ernst Haeckel. G. Fischer Verlag, Jena. WIESEN, B., KRUG, E., FIEDLER, K., WRAY, V,, and PROKSCH, P. 1994. Sequestration of hostplant derived flavonoids by the Lycaenid butterfly Polyommatus icarus. J. Chem, Ecol. 20:25232538. ZOPF, W, 1896. Flechtenstoffe. G, Fischer Verlag, Jena, ZUKAL. H. 1895. Morphotogische und biologische Untersuchungen fiber die Flechten. Sber. K. BOhm. Ges. Wiss. Math. -Nat. KI. 104:1303-1395.
