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Ecology, 82(7), 2001, pp. 2059–2071q 2001 by the Ecological Society of America

HOST–PARASITE–HERBIVORE INTERACTIONS:IMPLICATIONS OF HOST CYANOGENESIS

SUSANNA PUUSTINEN1,3 AND PIA MUTIKAINEN1,2

1Section of Ecology, Department of Biology, University of Turku, FIN-20014 Turku, Finland2Experimental Ecology, ETH-Zurich, ETH-Zentrum NW, CH-8092 Zurich, Switzerland

Abstract. To examine the mediation of host–predator interaction by a parasite, westudied the three-level interactions among a host plant, a root hemiparasitic plant, and theircommon predator, a generalist snail herbivore. The host species, Trifolium repens, is ableto synthesize cyanogenic glucosides that have a significant role in plant herbivore resistance.Some T. repens populations are polymorphic with respect to cyanogenesis. Our second aimwas to examine whether the mediation of the host plant–herbivore interaction by the parasiticplant could affect the maintenance of the cyanogenic polymorphism.

The parasitic plant (Rhinanthus serotinus) was equally harmful to cyanogenic and acyan-ogenic hosts and grew equally well on both host types. In a no-choice experiment, bothcyanogenesis and root hemiparasitism of the host plant reduced the growth of the herbivore(Arianta arbustorum). The herbivores consumed less leaf area of the parasitized plants thanof unparasitized plants, but only when the host plant was acyanogenic. In a multiple-choiceexperiment, the snails were similarly affected by cyanogenesis but not by parasitic infectionof the host. Thus, there was a discrepancy between food choice and performance of theherbivore. Our results suggest that the overall effects of the parasitic plant on the host plantwere ameliorated through the indirect effects of the parasitic infection on herbivore per-formance and food consumption. This indirect effect of the parasitic infection seemed tobe more beneficial for the acyanogenic plants: if parasitized, the leaf area of acyanogenicplants consumed by the herbivore was 45% higher than that of cyanogenic plants, whereasin unparasitized plants the corresponding figure was 114%. Thus, parasitism may decreasethe advantage cyanogenic plants gain through decreased herbivory. Further, in mainly cy-anogenic T. repens populations, cyanogenesis might be a more important feeding deterrentthan root parasitism; whereas in mainly acyanogenic populations root parasitism might berelevant to herbivore–T. repens interactions. We are the first to document the effects of aplant parasite on the performance of both the host plant and a host-feeding herbivore. Ourresults highlight the need to look beyond the direct effect of parasites on their hosts.

Key words: Arianta arbustorum; cyanogenesis; cyanogenic polymorphism; food choice; hemi-parasitic plants; host–parasite interactions; plant–herbivore interactions; Rhinanthus serotinus; Scro-phulariaceae; Trifolium repens.

INTRODUCTION

In many host–parasite interactions the effects of par-asitism extend beyond the direct negative effect of theparasites on host growth, reproduction, and survival.Parasites manipulate host behavior (Maitland 1994,Barber and Huntingford 1996, Jakobsen and Wedekind1998), affect host vulnerability to predation (Price etal. 1986, Lefcort and Blaustein 1995, Lafferty and Mor-ris 1996) and herbivory (Hammond and Hardy 1988,Ramsell and Paul 1990, Linhart 1991, Gomez 1994,Ericson and Wennstrom 1997), and shape the structureand dynamics of host communities by changing thecompetitive interactions among host species (Park1948, Price et al. 1986, Minchella and Scott 1991,Schall 1992, Bonsall and Hassel 1997, Van Der Puttenand Peters 1997, Hudson and Greenman 1998). In this

Manuscript received 27 September 1999; revised 14 June2000; accepted 28 June 2000; final version received 19 July 2000.

3 E-mail: [email protected]

respect, parasitic plants are no exception; they can me-diate the competitive interactions among their host spe-cies (Gibson and Watkinson 1992, Pennings and Cal-laway 1996, Davies et al. 1997). Further, parasiticplants can mediate interactions of their hosts with pol-linators and herbivores (Gomez 1994). Similarly, hostplants can influence interactions between parasiticplants and their herbivores; for example, by affectingthe nutritional quality of the parasite, which can affectthe susceptibility of the parasite to its own herbivores(Harvey 1966, Marvier 1996, 1998). The relative valueof different host species for the parasite can also dependon the host response to defoliation (Salonen and Puus-tinen 1996, Puustinen and Salonen 1999a). Previousstudies on parasitic mediation of host plant’s interac-tions with herbivores in natural systems have consid-ered how parasites and pathogens influence host sus-ceptibility to herbivory (e.g., Gomez 1994, Ericson andWennstrom 1997). We took a new approach to thisthree-level interaction; in addition to studying host sus-

2060 SUSANNA PUUSTINEN AND PIA MUTIKAINEN Ecology, Vol. 82, No. 7

ceptibility to herbivores, we examined how parasiticplants influence both their host plants and the perfor-mance of a host-feeding herbivore.

Many plant species produce secondary metabolitesthat have a role in plant resistance and toxicity to her-bivores (Harborne 1997, Karban and Baldwin 1997).We used white clover, Trifolium repens, as the hostspecies since it is polymorphic with respect to cya-nogenesis, with both cyanogenic and acyanogenic in-dividuals occurring in the same populations (Harborne1988, Kakes 1989). In white clover, the cyanogenicmorph is often more frequent in low latitudes and al-titudes (Daday 1954a, b, Till 1987, Till-Bottraud et al.1988, Ganders 1990). Many herbivore species, espe-cially snails and slugs, prefer acyanogenic plants overthe cyanogenic plants, and cyanogenic individuals suf-fer less damage due to selective feeding (Jones 1962,1966, Crawford-Sidebotham 1972, Angseesing 1974,Dirzo and Harper 1982a, b, Compton et al. 1983, Hor-rill and Richards 1985, Mowat and Shakeel 1988,Saucy et al. 1999). On the other hand, compared toacyanogenic individuals, cyanogenic individuals havebeen suggested to be less tolerant of drought, frostdamage, and pathogenic fungi (Foulds and Grime1972a, b, Dirzo and Harper 1982b, Till-Bottraud andGouyon 1992). Thus, the cyanogenic polymorphismseems to be maintained by selection by herbivores forthe cyanogenic and against the acyanogenic morph, andby selection by temperature against the cyanogenic andfor the acyanogenic morph (Foulds and Grime 1972b,Dirzo and Harper 1982a, b; for a review, see Hughes1991).

Parasitic plants may affect the cyanogenic polymor-phism of their host in two ways. First, parasitic plantsmay have stronger negative direct effects on the cya-nogenic hosts compared to the acyanogenic ones, pro-viding a selective advantage to the acyanogenic morph.White clover genotypes that contain cyanogenic glu-cosides produce fewer flowers than the acyanogenicgenotypes, suggesting that cyanogenesis is costly interms of reproductive output (Foulds and Grime 1972b,Dirzo and Harper 1982a, b, Caradus et al. 1989, Kakes1989, 1997). The costs of cyanogenesis might be fur-ther expressed as reduced tolerance to biological en-emies other than herbivores, such as parasitic plants orpathogens (Dirzo and Harper 1982a, b). Thus, acyan-ogenic individuals may have more resources than cy-anogenic individuals for compensating for damagecaused by parasites or pathogens. Second, a parasiticplant may indirectly affect host-feeding herbivores bychanging the nutritional quality of host leaves. If theseindirect effects differ between the cyanogenic andacyanogenic morph, they might affect the cyanogenicpolymorphism.

We used an experimental approach to study the three-level interactions among a host plant, Trifolium repens,a parasitic plant, Rhinanthus serotinus, and their sharedgeneralist herbivore, the snail Arianta arbustorum.

First, we examined the direct effects of host cyano-genesis on the host–parasite interaction. Second, weexamined whether the effects of host cyanogenesis arereflected in the host–herbivore and parasite–herbivoreinteraction. We addressed three specific questions con-cerning the host–parasite interaction. (1) Does host cy-anogenesis affect the ability of the host plant to toleratethe parasitic infection? (2) Do root parasitic plants af-fect the nutrient levels of cyanogenic and acyanogenichosts? (3) Does the parasitic plant perform equally wellon acyanogenic and cyanogenic host plants? We ad-dressed two specific questions concerning the plant–herbivore interactions. (1) Can a generalist herbivorechoose simultaneously against the cyanogenic and par-asitized food, and is the food choice reflected in her-bivore performance? (2) Is a generalist herbivore ableto select between hemiparasitic plants grown on cya-nogenic and acyanogenic hosts, and is the putative foodchoice further reflected in herbivore performance?

METHODS

Natural history

The host plant, white clover (Trifolium repens), is aperennial, clonal plant with nitrogen-fixing root sym-bionts. T. repens is a cosmopolitan species that growson roadsides and natural grasslands in Finland. Cya-nogenic individuals synthesize cyanogenic glucosidesthat are stored separately from their hydrolytic enzymes(Hughes 1991). Both the enzymes and the cyanogenicglucosides are always present in the leaves of cyano-genic individuals but hydrogen cyanide (HCN) is re-leased only when the tissues are damaged and the hy-drolytic enzymes come into contact with the cyano-genic glucosides (Harborne 1988). The expression ofcyanogenesis in T. repens is controlled by two inde-pendent loci. One of the loci (Ac/ac) controls the pro-duction of cyanogenic glucosides (linamarin and lo-taustralin) while the other locus (Li/li) controls the pro-duction of an enzyme (linamarase) that hydrolyzes thecyanogenic glucosides (Corkill 1952, Hughes 1991).Only the genotype that possesses dominant alleles inboth loci produces both the cyanogenic glucoside lin-amarin and the enzyme linamarase (i.e., Ac Li) andreleases HCN when leaves are damaged. Hydrogen cy-anide is a moderately toxic substance to herbivores dueto its ability to combine with enzymes associated withcellular respiration, e.g., cytochrome oxidase (Conn1979). Compounds other than HCN can also partly af-fect the toxicity of cyanogenic plants (Seigler 1991).

Approximately one percent of all angiosperm speciesare parasitic on other plants (Atsatt 1983). Root andshoot parasites attach to belowground or abovegroundparts of their hosts, respectively (Musselman and Press1995). Parasitic plants are also classified as hemi- andholoparasites, depending on the presence or absence ofchlorophyll, respectively (Musselman and Press 1995).Parasitic plants are common components in many

July 2001 2061HOST–PARASITE–HERBIVORE INTERACTIONS

PLATE 1. (a) Rhinanthus serotinus is a common component of roadside plant communities where it parasitizes manyleguminous and grass species (photograph by Tanja Koskela). (b) In Finland, Rhinanthus serotinus flowers from June untilthe end of August (photograph by Leena Lindstrom). (c) Arianta arbustorum is a generalist herbivore snail that occurs innutrient-rich habitats of southern Finland. It can occur locally in high densities (photograph by Tanja Koskela).

grassland communities from the arctic to the tropics(Musselman and Press 1995). The extraction of water,nutrients, and carbon by root hemiparasitic plants fromthe host xylem via haustorial root connections can sub-stantially reduce growth and reproduction of the hostplant (Govier et al. 1967, Press et al. 1987, 1988, 1990,Gibson and Watkinson 1991, Cechin and Press 1994,Clark et al. 1994, Graves 1995, Marvier 1996, Puus-tinen and Salonen 1999a, b). The largest number ofroot hemiparasites occurs in the family Scrophulari-aceae (Atsatt 1983). Most root hemiparasitic plants aregeneralists with respect to host plant species. The par-asite we used, Rhinanthus serotinus (Scrophulari-aceae), is a partially autotrophic, generalist root par-asite (Klaren and Van de Dijk 1976, Klaren and Janssen1978). Rhinanthus serotinus is widespread in Europe(Polunin 1969), and grows in natural and roadsidegrasslands. Nitrogen-fixing plants, such as T. repens,are often considered the most beneficial hosts for theroot hemiparasites in the family Scrophulariaceae be-cause of their high nitrogen levels (Gibson and Wat-kinson 1991, Seel and Press 1994).

The herbivore used in this study, Arianta arbustorum(Helicidae), is a hermaphroditic snail that occursthroughout Central, Eastern, and Northern Europe (Ter-hivuo 1978, Burla and Stahel 1983, Kerney et al. 1983).Arianta arbustorum is a generalist herbivore (From-ming 1937, Speiser and Rowell-Rahier 1991) that pre-fers habitats with rich and dense vegetation (Terhivuo1978). It feeds on living and dead plant material aswell as on wilted flowers, mushrooms, arthropods, andsoil (Fromming 1937, Speiser and Rowell-Rahier1991). In southern Finland, A. arbustorum becomes

reproductively mature during its third or fourth summer(Terhivuo 1978).

Rearing of plants and herbivores

We collected the seeds of the parasite, Rhinathusserotinus, at the end of September 1996, from morethan 100 plants growing in a roadside grassland in Kon-nevesi, Central Finland (628379 N, 268219 E). The seedswere stored in paper bags at room temperature for fivemonths and then incubated between moist filter paperat 48C for two months, after which the first seeds startedto germinate. Seeds of the host plant, Trifolium repens,were provided by the Welsh Plant Breeding Station,Aberystwyth, Ceredigion, Wales. The acyanogenic andcyanogenic lines that we used were originally devel-oped from the same variety (AC52), and are, therefore,morphologically very similar. The frequency of HCNpositive plants is 17% in the acyanogenic line (AC52B)and 94% in the cyanogenic line (AC52D).

Immediately after the parasite seeds started to ger-minate, a large number of host seeds were germinatedon wet paper in Petri dishes. The host seeds germinatedwithin 24 h. The host seedlings were then transplantedto pots filled with a 1:1 mixture of fertilized soil (Bio-lan, Puutarhan Musta Multa; Biolan Oy, Kauttua, Fin-land) and fine-grained sand. Ten days later, we trans-planted the host seedlings (2 cm tall with three leaves)individually to 0.5 L pots. We inoculated all pots withthe nitrogen-fixing symbiont of clover, Rhizobium leg-uminosarum biovar. trifolii (HAMBI 461), to ensurethat all experimental plants had symbiotic bacteria. TheRhizobium were mixed into pulverized, fine-grainedpeat (10% activated carbon and 6% CaCO3) at a con-


centration of 910 bacteria per gram of peat; 2.5 g of thisbacteria mixture was added to 3 L of water, and 5 mLwas added to each pot.

We transplanted three parasite seedlings (radicalstage) per host into half of the pots; half of the hostsgrew without the parasites. We also established a treat-ment in which the parasitic plants grew without thehost plant. After the emergence of cotyledons wethinned the parasite seedlings to one per pot. The potswere randomly arranged on greenhouse benches androtated weekly during the experiment. The temperatureinside the greenhouse followed the outside tempera-ture. The plants were watered when needed and nofertilizers were applied. The plants used in the follow-ing experiments were randomly selected from theseplants and each plant was used only once.

Before each of the experiments, the host plants werescored for their ability to produce HCN using a sodiumpicrate test (see Corkill 1940, Jones 1966, Dirzo andHarper 1982a for methods). We placed two leaves (in-cluding the youngest leaf) of each plant in a test tube,added two drops of toluene, and then crushed the leafmaterial with a glass rod. Strips of filter paper that hadbeen soaked in a solution of sodium picrate were thensuspended in the corked test tubes. The test tubes wereincubated at 378C for 24 h. If the paper strip changedits color from the original yellow to red or brown, theplant was scored as cyanogenic. We used only clearlyacyanogenic plants from line AC52B and clearly cy-anogenic plants from the line AC52D for the experi-ments. The sodium picrate test only separates the cy-anogenic (Ac Li) plants from the three acyanogenictypes (i.e., Ac li, ac Li, and ac li). The number ofreplicates among treatments within experiments variesfor two reasons. First, some plants in both the acyan-ogenic line and the cyanogenic line were not acyano-genic or cyanogenic, respectively, and were excludedfrom the analyses. Second, some of the parasites diedbefore the end of the experiments. The number of rep-licates is shown in figures.

We collected both juvenile and mature A. arbustorumsnails in July and August 1997 from Turku (60859 N,228299 E) for the growth and food-choice experiments.Prior to the experiments, the adult snails were keptindividually in Petri dishes (13.5 cm in diameter) andthe juvenile snails were kept in plastic buckets, and fedwith fresh lettuce. Before and during the experiments,the snails were kept in a dark room at 178C. Maturesnails did not produce eggs during the experiments.During the experiments, the snails were kept on moistfilter paper in Petri dishes. Snail feces were removedfrom the dishes regularly. We assigned one food plantfor each snail and each snail was used only once. Plantsused for the experiments were 11–12 wk old. In allexperiments, we measured the leaf area eaten by pho-tocopying the leaves before and after the leaves wereoffered to snails. Leaf areas were analyzed using an

image analysis system (MCID, M4, Image Research,Belfast, Northern Ireland).

Host–parasite interaction

To study the effects of host cyanogenesis and par-asitism on the performance of parasites and hosts, weconducted a fully factorial experiment with host cya-nogenesis (acyanogenic/cyanogenic) and parasitic in-fection (yes/no) as factors. In an additional treatment,the parasitic plants grew without host plants. At theend of the 11-wk experiment, we counted the numberof flowers produced by the host and by the parasite.The roots of the plants were washed, and the parasiticplants and host plants were carefully separated fromeach other. All plant parts were dried (1008C, 48 h)and weighed. The data on total biomass of the hostplant were statistically tested with a two-way analysisof variance (ANOVA). The data on total biomass andflower number of the parasitic plant were tested withone-way MANOVA, and pairwise comparisons wereconducted using Tukey’s hsd test. All statistical testswere performed with SPSS for Windows (Norusis1993).

In addition to the direct toxic effects of host plantcyanogenesis, the food choice and growth of herbivoresfeeding on the host plant may have been determinedby changes in nutrient concentrations of the host plantdue to cyanogenesis and parasitic infection. To examinethe effects of host cyanogenesis and hemiparasitism onthe nutrient concentrations of the host leaves, 10 ran-domly selected host plants per treatment were analyzedfor leaf nitrogen, phosphorus, calcium, magnesium, andpotassium concentrations in Novalab Oy (Karkkila,Finland). The data on nutrient concentrations were an-alyzed with a two-way multivariate analysis of variance(MANOVA).

Plant–herbivore interactions: parasitism,cyanogenesis, and host-feeding herbivore

Growth of snails.—To study the effects of host plantparasitism and cyanogenesis on growth and food choiceof the snails, we used a fully factorial experimentaldesign with host cyanogenesis (acyanogenic/cyano-genic) and parasitic infection (yes/no) as factors. In thegrowth experiment, we randomly assigned juvenilesnails (mass 0.05 6 0.0030 g, mean 6 1 SE) to fourhost plant groups according to the factorial design. Wereplaced host leaves every second day with freshleaves. The snails were weighed before and after the14-d experiment. The relative growth of the snails wascalculated as (we 2 wb)/wb, where we and wb are snailbiomass at the end and in the beginning of the exper-iment, respectively. The relative growth of the snailsand the total area of leaves consumed during the ex-periment were analyzed with two-way MANOVA.

Food choice of snails.—We studied the effects ofhost plant parasitism and cyanogenesis on snail foodchoice in a multiple-choice experiment where the four


FIG. 1. Effects of host cyanogenesis and parasitism bythe root hemiparasitic plant (Rhinanthus serotinus) on themean total biomass (1 1 SE) of the host plant (Trifoliumrepens). Numbers of replicates are shown inside the bars.

FIG. 2. Effects of host plant (Trifolium repens) cyano-genesis on (A) the mean total biomass (1 1 SE), and (B)number of flowers of the root hemiparasitic plant (Rhinanthusserotinus). Bars marked with the same letter do not differsignificantly at the P , 0.05 level (Tukey’s test).

types of host leaves (acyanogenic parasitized, cyano-genic parasitized, acyanogenic unparasitized, cyano-genic unparasitized) were offered simultaneously. Weconducted the experiments separately for juvenile (0.056 0.0046 g) and adult snails (1.64 6 0.2 g). In thechoice tests, the snails were offered similar-sized leavesthat were placed in symmetric circular form on thebottom of Petri dishes to give the snails an equal choiceamong the leaves. We randomly assigned one host plantfrom each of the four treatment groups to one snailindividual. The juvenile snails were simultaneously of-fered one leaf from each treatment group (i.e., the totalnumber of leaves offered per Petri dish was four) andthe adult snails were offered two leaves from each treat-ment group (i.e., the total number of leaves offered waseight). The food-choice experiment lasted 24 h for adultand 48 h for juvenile snails. We measured leaf areasbefore and after the choice experiments. We alsoweighed the leaves offered to the adult snails beforeand after the experiment. The data were analyzed withrepeated-measures ANOVA having the cyanogenesisand parasitism as the within-effects factors.

Plant–herbivore interactions: cyanogenesis andparasite-feeding herbivore

Growth of snails.—To study the effects of host plantcyanogenesis on herbivore growth on parasite leaveswe fed juvenile snails (0.05 6 0.0049 g) with leavesof parasites that grew either on acyanogenic or cya-nogenic hosts. Otherwise, we conducted this experi-ment similarly to the growth experiment with the hostleaves (see Methods: Plant–herbivore interactions:parasitism, cyanogenesis, and host-feeding herbivore:subsection Growth of snails). The relative growth ofthe snails and the leaf area eaten during the experimentwere analyzed with t tests.

Food choice of snails.—In this experiment, juvenile(0.08 6 0.01 g) and adult (1.15 6 0.03 g) snails chosebetween the leaves of parasites that grew either onacyanogenic or on cyanogenic hosts. The leaves wereplaced in a symmetric circular form on the bottom ofthe Petri dish to give the snails an equal choice. Fourleaf pieces (0.5 cm 3 2 cm) per treatment (together

eight pieces) were offered for each snail. The experi-ment lasted 24 h and 48 h for the adult and juvenilesnails, respectively. We measured the leaf area eatensimilarly to the previous experiments, and analyzed thedata with a pairwise t test.

Uptake of HCN from the host plant by theparasitic plant

Some parasitic plants are able to extract defensivecompounds, such as alkaloids, from their hosts (e.g.,Stermiz et al. 1989, Scheider and Stermiz 1990, MartınCordero et al. 1993, Marvier 1996, Marko and Stermiz1997). This way, parasitic plants may use the defensivecompounds of their hosts for protection against her-bivores. However, it is unlikely that root parasites couldgain a direct advantage from host cyanogenesis; HCNis only released when the leaves are damaged becausecyanogenic glucosides and the hydrolytic enzymes areseparated by compartmentation within the leaf (e.g.,Jones 1972, Conn 1979, Seigler 1991).

To exclude the possibility of HCN production by theparasitic plant or the movement of HCN from hosts toparasites, we conducted the sodium picrate tests (seeCorkill 1940, Jones 1966, Dirzo and Harper 1982a formethods) on the leaves of 40 randomly selected par-asitic plants. We checked the production of HCN bythe parasite when the leaves of the host plant wereeither not crushed or crushed to induce HCN. In thefirst treatment (N 5 20), the cyanogenic hosts and theirparasites grew for nine weeks after which we conducteda sodium picrate test to measure cyanogenesis in theparasite and host leaves. In the second treatment (N 5


TABLE 1. Results of MANOVA for the effects of host (Trifolium repens) cyanogenesis (C) and parasitism by Rhinanthusserotinus (P) on the leaf nitrogen, calcium, phosphorus, potassium, and magnesium concentration (g/kg dry mass) of the host.

Source

MANOVA (Wilks’ lambda)

df F P

Nitrogen

MS F P

Calcium

MS F P

CPC 3 PError

111

32

5.310.981.07

0.0010.4460.398

4.2953.59

0.1813.66

0.313.920.01

0.5790.0550.909

230.401.44

29.9312.18

18.920.122.46

,0.00010.7330.126

Note: Error df 5 36 for the univariate analyses.

20), the host plants and their parasites grew for nineweeks after which we crushed three leaves of the hostsbetween two small stones once in every hour during10 h. After the crushing, we conducted sodium picratetests for the parasite and host leaves. We repeated thesodium picrate test 12 h after the first test was madefor the parasitic plants. We did not detect any cyano-genesis in the leaves of parasites, either when hostleaves were crushed or when they were not crushed.

RESULTS

Host–parasite interaction

Performance of the host.—Parasitic infection re-duced the total biomass of the host by 57% (ANOVA:F1,47 5 102.52, P , 0.0001; Fig. 1). Host cyanogenesisdid not significantly affect host biomass (F1,47 5 1.03,P 5 0.315), and there was no statistically significantinteraction between parasitic infection and host cya-nogenesis (F1,47 5 0.61, P 5 0.440). The results weresimilar for host root and shoot biomass (data notshown). Only five host plants flowered during the ex-periment. The results did not differ qualitatively if theflowering individuals were excluded from the analyses.

Performance of the parasite.—Host plant treatment(no host/acyanogenic host/cyanogenic host) signifi-cantly affected the total biomass of the parasite, as wellas the number of flowers produced (MANOVA: Wilks’lambda F4,50 5 18.09, P , 0.0001; total biomass: F2,26

5 51.84, P , 0.0001; number of flowers: F2,26 515.044, P , 0.0001; Fig. 2). The parasites that grewwithout hosts had significantly lower total biomass andnumber of flowers when compared to the other twohost treatments (Tukey’s test, Fig. 2). There were nosignificant differences in these traits between parasitesgrown on acyanogenic host and parasites grown oncyanogenic hosts (Tukey’s test, Fig. 2). The proportionof the parasite biomass to the total plant biomass didnot differ significantly between pots with acyanogenichosts (0.45 6 0.04) and pots with cyanogenic host (0.396 0.05; t test, t15 5 0.84, P 5 0.413). The results forthe root and shoot biomass of the parasite were similarto those for the total biomass and flower numbers (datanot shown).

Host nutrient concentrations.—Parasitism tended todecrease the nitrogen concentration of host leaves (P5 0.055) but did not affect the concentration of any

other nutrient that we measured (Table 1). There wereno significant differences in the leaf nitrogen, phos-phorus, or potassium concentrations between acyano-genic and cyanogenic hosts (Table 1, Fig. 3A, B). How-ever, the calcium concentration of acyanogenic hostswas higher than that of cyanogenic hosts (Table 1, Fig.3A). There was a significant interaction between par-asitism and cyanogenesis for leaf magnesium concen-tration; the magnesium concentration tended to be low-er in the cyanogenic hosts than in the acyanogenic hosts(P 5 0.083, Table 1); however, this difference wassignificant only in the unparasitized plants (t test, t18

5 2.85, P 5 0.011 for unparasitized plants, t18 5 20.45,P 5 0.658 for parasitized plants, Fig. 3B.)

Plant–herbivore interactions: parasitism,cyanogenesis, and host-feeding herbivore

Growth of snails.—The relative growth rate of ju-venile snails was significantly lower on leaves of par-asitized hosts than on leaves of unparasitized hosts (Ta-ble 2, Fig. 4A). Further, snails fed with cyanogenichosts grew slower than those fed with acyanogenichosts (Table 2, Fig. 4A). Correspondingly, snails thatwere fed with leaves of cyanogenic or parasitized hostsate less (measured as mm2) than snails that were fedwith leaves of acyanogenic or unparasitized hosts, re-spectively (Table 2, Fig. 4B). We also found a statis-tically significant interaction between hemiparasitic in-fection and food plant cyanogenesis in their effects onthe leaf area eaten (Table 2). Snails fed with cyanogenichosts consumed similar amounts of leaves from para-sitized and unparasitized hosts (t test, t62 5 0.09, P 50.92); whereas, snails fed with acyanogenic hosts con-sumed more leaves of unparasitized than of parasitizedhosts (t61 5 2.738, P 5 0.008, Fig. 4B).

Food choice of snails.—When food choice of thesnails was measured as leaf area eaten in the multiple-choice experiment, both juvenile and adults snails pre-ferred the leaves of acyanogenic host plants over thoseof cyanogenic host plants although the difference wasonly marginally significant (Table 3, Fig. 5). In addi-tion, adult snails preferred acyanogenic leaves over cy-anogenic ones when food choice was measured as freshleaf biomass eaten in grams (Table 3, Fig. 5C). Parasiticinfection of the host plant did not affect the food choiceof adult or juvenile snails (Table 3, Fig. 5); thus, the


TABLE 1. Extended.

Phosphorus

MS F P

Potassium

MS F P

Magnesium

MS F P

0.110.090.020.20

0.550.460.09

0.4620.5000.765

38.420.96

21.6112.11

3.170.081.78

0.0830.7800.190

1.500.00442.700.47

3.180.015.74

0.0830.9230.022

FIG. 3. Nutrient concentrations (1 1 SE) of Trifolium re-pens leaves at the end of the experiment. C 5 cyanogenesis,P 5 parasitism; ‘‘1’’ denotes presence, and ‘‘2’’ denotesabsence.

snails did not discriminate against the parasitized foodplants, even if they grew significantly less well onthem.

Plant–herbivore interactions: cyanogenesis andparasite-feeding herbivores

Growth of snails.—The relative growth of juvenilesnails was negative when they were fed with the leavesof the parasitic plant, regardless of whether the parasitewas growing on acyanogenic or cyanogenic host(20.17 6 0.22 and 20.16 6 0.20, respectively; t test,t44 5 20.17, P 5 0.866). Correspondingly, the leaf areaconsumed by the snails did not differ between parasitesthat grew on acyanogenic hosts and parasites that grewon cyanogenic hosts (311 6 40 mm2 and 264 6 38mm2, respectively; t test, t44 5 0.857, P 5 0.396). It isnotable that the snails consumed the leaves of the par-asitic plants to the same degree as they consumed the

leaves of the host plants (in terms of leaf area eaten,see Fig. 4B). Thus, the negative growth is not explainedby very low food consumption during the experiment.

Food choice of snails.—Neither juvenile nor adultsnails showed any preference between the leaves ofparasites that grew on acyanogenic hosts and parasitesthat grew on cyanogenic hosts in the food-choice ex-periments (pairwise t test, juveniles: t29 5 0.69, P 50.496; adults: t42 5 20.47, P 5 0.643, Fig. 6).

DISCUSSION

Parasitic mediation of host–herbivore interaction

In this experiment, parasitism significantly reducedhost biomass, in accordance with previous experimentswhere root parasitism has been observed to reduce hostbiomass and reproduction (Matthies 1995a, b, Puus-tinen and Salonen 1999a, b). On average, parasitic in-fection decreased host total biomass by 57%, and wasequally harmful to both cyanogenic and acyanogenichost plants. However, the parasitic infection mediatedhost–herbivore interaction in three ways. First, the per-formance of a host-feeding generalist herbivore wasaffected by the parasitic infection of the host; the her-bivores feeding on the leaves of a parasitized host plantgrew less (52%) when compared to those feeding onthe leaves of unparasitized hosts, regardless of the hosttype. Second, the food consumption of herbivores feed-ing on parasitized hosts was lower (on average 24%)than that of herbivores feeding on unparasitized hosts.Third, there was a discrepancy between the food choiceand performance of the herbivore; even if snail growthwas delayed on leaves of parasitized hosts plants, theywere not able to discriminate against this food in amultiple-choice experiment, especially if the host wascyanogenic. Together these three results indicate thatthe overall effects of the parasitic infection may beameliorated by the effects of the infection on host–herbivore interaction. As previously emphasized instudies on other host–parasite interactions, our resultshighlight the need to look beyond the two-way inter-action when measuring the effects of parasites on theirhosts.

We are the first to document the indirect negativeeffects of parasitic plants on the performance of host-feeding herbivores. A previous study on the mediationof host plant–herbivore interaction by parasitic plantsconcentrated on the changes in host plant susceptibility


TABLE 2. Results of MANOVA for the effects of host (Trifolium repens) cyanogenesis (C)and parasitism (P) on the growth rate of host-feeding snails (juvenile Arianta arbustorum)and on the amount of host leaf area eaten (mm2) by the snails during the experiment.

Source

MANOVA(Wilks’ lambda)

df F P

Relative growth rate

MS F P

Leaf area eaten

MS F P

CPC 3 PError

111

122

22.265.903.01

,0.00010.0040.053

0.640.200.040.03

21.766.661.21

,0.0010.0110.273

1 156 688.5263 292.4239 900.6

48 744.4

23.7305.4014.922

,0.00010.0220.028

Note: Error df 5 123 for the univariate analyses.

FIG. 4. Effects of host plant hemiparasitism and cyano-genesis on (A) the relative growth, and (B) the amount ofleaf area eaten (1 1 SE) by juvenile Arianta arbustorum snails.Numbers of replicates are shown inside the bars.

to herbivores (Gomez 1994). The shoot parasite dodder,Cuscuta epithymum (Cuscutaceae), decreased the pol-linator and herbivore visitation rates of its host, thewoody shrub Hormatophylla spinosa (L.) Kupfer L.(Cruciferae). Interestingly, after both the direct nega-tive effect of the parasite on host performance and theindirect effects via pollinators and herbivores were tak-en into account, the overall influence of the parasiticplant on the host plant was negligible (Gomez 1994).With respect to other plant pathogens and in contrastto our results, the pathogenic rust fungus, Urocystistrientalis L., enhanced herbivory by voles and scaleinsects on Trientalis europaea L. in natural conditions(Ericson and Wennstrom 1997).

The parasitic infection did not have any implicationsfor the maintenance of the cyanogenic polymorphismthrough its direct effects; the effects of parasitism weresimilar on cyanogenic and acyanogenic hosts. How-

ever, the indirect effects of parasitism differed betweenthe cyanogenic and acyanogenic host in one importantrespect. In the growth rate experiment (no-choice test),the herbivores consumed less leaf area of the parasit-ized plants, but only when the host plant was acyan-ogenic. This result suggests that when parasitized, thedifference in the amount of herbivory between cya-nogenic and acyanogenic plants may be smaller com-pared to the situation when the plants are not parasit-ized. This indirect effect of parasitism may partly favoracyanogenic plants in habitats where both herbivoresand parasitic plants are present.

Herbivore performance and food choice

Parasitism of T. repens did not affect the food choiceof A. arbustorum snails, although it decreased the ni-trogen concentrations of both the cyanogenic andacyanogenic host plants, and also reduced the growthof the snails. There are several explanations for thisdiscrepancy. First, the nutritional value of food plantsprobably contributes to the patterns of food consump-tion by A. arbustorum in the field (Speiser and Rowell-Rahier 1991). In this study, the reduction in nitrogenconcentration of host leaves caused by the parasite was.10%. It is possible that in the multiple-choice ex-periment this reduction in the nitrogen concentrationsimply was not a strong enough signal to cause dis-crimination against parasitized host plants. Second,there were differences in the duration of the experi-ments; the choice tests lasted for only one or two days,whereas the growth rate experiment lasted for twoweeks. The food selectivity of slugs has been shownto increase with time; i.e., with familiarity with thefood items under selection (Angseesing 1974). Thus,in the multiple-choice experiment the snails may nothave had enough time to physiologically respond to thefood plant, which then would have led to discriminationagainst the parasitized food plant. Finally, cyanogen-esis functions as a deterrent and toxin (e.g., Conn 1979,Dirzo and Harper 1982a, Seigler 1991), and a strongsignal probably prohibited the snails from respondingsimultaneously to parasitism. If the food-choice ex-periments had been conducted as paired-choice tests,the snails may have been able to discriminate againstparasitized host plants, especially if the host plants


TABLE 3. Results of repeated-measures ANOVA for the effects of host (Trifolium repens)cyanogenesis (C) and parasitism (P) on the food choice of the host-feeding snails (Ariantaarbustorum).

Source

Juveniles

Leaf area eaten

MS F P

Adults

Leaf biomass eaten

MS F P

Leaf area eaten

MS F P

CPC 3 P

1.901.160.75

4.121.981.62

0.0510.1690.212

0.0010760.0000720.000154

4.950.430.70

0.0320.5180.408

25318.081251.761253.58

3.420.220.21

0.0720.6430.648

Notes: Food choice of juvenile snails was measured as leaf area eaten (mm2), and that ofadult snails was measured as leaf biomass (g) and leaf area (mm2) eaten. Error df 5 31 forjuveniles and 39 for adults.

FIG. 6. Effect of host plant cyanogenesis on food choice(1 1 SE) of (A) hemiparasite-feeding juvenile and (B) adultArianta arbustorum snails. Numbers of replicates are showninside the bars.

FIG. 5. Effects of host plant hemiparasitism and cyano-genesis on the food choice (1 1 SE) of (A) juvenile and (B,C) adult Arianta arbustorum snails. Numbers of replicates areshown inside the bars.

were acyanogenic. This suggestion is supported by theresults of the no-choice growth experiment in whichthe snails ate less of the parasitized acyanogenic foodplants than unparasitized acyanogenic food plants.Based on the results of these laboratory experiments,cyanogenic food plants may have at least two disad-vantages to snails. First, cyanogenic food plants arepoisonous and snails grow poorly if they feed on them.

Second, cyanogenesis hinders the snails from discrim-inating against food items that have a lower nutritionalvalue for other reasons (such as the parasitic infectionin this study).

There are at least three reasons why herbivores preferacyanogenic food plants. First, HCN is a toxic com-pound as such. For example, Arianta arbustorum pre-ferred petals of acyanogenic Lotus corniculatus plantsover those of cyanogenic plants (Compton and Jones1985), and a slug, Deroceras reticulatum, preferred tograze on the acyanogenic morph of T. repens over thecyanogenic morph (Burgess and Ennos 1987). How-ever, some herbivorous mollusk species do not exhibita preference between cyanogenic or acyanogenic plants(Crawford-Sidebotham 1972, Angseesing 1974). Sec-ondly, cyanide is a nitrogen-containing compound;Thus, cyanogenic plants, although they have the same


total nitrogen concentration as acyanogenic plants, mayhave lower levels of nitrogen available to the herbivorebecause part of the nitrogen is contained in cyanide.Thirdly, in this experiment, acyanogenic plants had ahigher leaf calcium concentration than the cyanogenicones. The calcium concentration may have additionallystimulated the snail feeding because calcium is an es-sential mineral element for snail shell growth (Godan1983, Graveland and van der Wal 1996).

Our results show that snails performed best when fedwith the acyanogenic food that they preferred in themultiple-choice experiment. Dirzo and Harper (1982a)have shown that the slugs (Agriolimax carunae) main-tained on cyanogenic T. repens had only slightly re-duced growth rates compared to slugs maintained onan acyanogenic diet. However, similar to this experi-ment, the lower growth rate in A. carunae might havebeen due to the lower consumption of cyanogenicleaves, rather than poisoning. In fact, it has been sug-gested that cyanogenesis primarily functions as a feed-ing inhibitor, rather than as a toxin (Compton and Jones1985).

Direct effects of parasitism on host performance

We expected that the uptake of nutrients and waterfrom the host plant by the parasitic plant would stressthe cyanogenic hosts more because cyanogenic plantsalso have to allocate resources to cyanogenesis. How-ever, our results do not support this expectation; par-asitic plants were equally harmful to cyanogenic andacyanogenic hosts when host performance was mea-sured as the biomass produced. There are several pos-sible explanations for this result. First, cyanogenesishas been shown to be costly in terms of flower pro-duction (Foulds and Grime 1972b, Caradus et al. 1989,Kakes 1989, 1997); but, to our knowledge, the costsof cyanogenesis have not been observed in terms ofvegetative growth. Only five plants flowered during thisexperiment; thus, the costs of cyanogenesis may nothave been expressed in the sexually immature plants.Second, in some cases, the costs of herbivore defensemay depend on resource availability, and costs are morelikely to be expressed in resource-limiting conditions(Bergelson 1994a, b). In this experiment, the plantsgrew under favorable conditions, probably without sig-nificant resource limitation. However, the parasitic in-fection decreased host biomass by 57% and it is highlylikely that this kind of damage would lead to decreasedreproductive success. Third, some studies have sug-gested that drought is more costly to acyanogenicplants than to cyanogenic plants. This has been basedon the suggestion that drought might induce the releaseof HCN by disturbing the water balance of the cells,thereby bringing the glucosides and enzymes into con-tact (Foulds and Grime 1972b). However, to our knowl-edge, there is no clear evidence for induction of HCNby drought. In theory, the extraction of water from thehost by the parasitic plant might also cause serious

water stress to the host, similar to drought, and inducethe release of HCN. However, in this experiment, wedid not observe any damage caused by water stress.Fourth, it should also be noted that preformed defenses,such as cyanogenesis, have been thought to be rela-tively inexpensive in the absence of herbivores whencompared to many other types of defenses (Karban andBaldwin 1997). Although there are costs to maintainthe defense system, a significant proportion of the costswill be expressed only when the defense is induced(Karban and Baldwin 1997). Therefore, parasitism mayhave had a stronger effect if cyanogenesis had beensimultaneously induced. Thus, in this laboratory ex-periment, the ability to produce HCN seems not to becostly for T. repens in terms of tolerance of a roothemiparasite. However, the effects of parasitism mightbe more severe in natural conditions where the hostsimultaneously suffers from drought, frost, resourcelimitation, disease, or herbivory.

Effects of cyanogenesis on the parasite andparasite-feeding herbivore

Our results indicate that the hosts’ ability to producecyanide had no significant effects on the reproductionof the parasitic plant in greenhouse conditions wherehost plants were not otherwise stressed. The lack of adifferential response might also be due to the fact thatthe costs of cyanogenesis were not expressed; there-fore, acyanogenic and cyanogenic plants were equallysuitable as hosts. The autotrophically grown parasiteshad 80% and 75% less biomass and produced 81% and79% fewer flowers than parasites grown with acyan-ogenic and cyanogenic host plants, respectively. In ac-cordance with this, several previous studies have shownthat the performance of generalist root hemiparasitesis substantially improved when they have access to ahost plant (Klaren and Van de Dijk 1976, Klaren andJanssen 1978, Seel and Press 1993, Matthies 1997).

The hemiparasitic plant was not a beneficial foodsource for A. arbustorum snails because the juvenilesnails lost mass during the growth experiment irre-spective of the cyanogenic nature of the host. SomeRhinanthus species contain aucubin-type glycosides(Hegnauer 1973, Gibbs 1974) that may make them un-palatable or toxic to herbivores. The results of the pre-sent experiment also indicate that root parasites do notextract HCN from the host plant; thus, host cyanogen-esis does not benefit the parasitic plant through its ef-fects on parasite-feeding herbivores or cost the para-sitic plant in terms of taking up a toxin. Usually le-gumes are nutrient rich, and the connected parasiticplants may be more susceptible to herbivory than par-asites connected to hosts with lower nutrient concen-trations. In accordance with this, aphids using hemi-parasites of legumes as food produced more offspringthan aphids using hemiparasites of nonleguminousplants as food (Marvier 1996). This might also makehemiparasites of legumes more susceptible to aphids


under natural conditions (Marvier 1996). According tothe results of Marvier (1996), the parasites also ex-tracted alkaloids from their hosts. Thus, from the par-asite-feeding herbivore’s viewpoint, the benefit of highnitrogen levels extracted from the leguminous host bythe parasite outweighed the detrimental effects of theextracted secondary compounds.

Conclusions

Since this study was conducted in laboratory andgreenhouse, we can not draw far-reaching conclusionsof the relevance of these results as explanations of pat-terns that exist in the field. However, we believe thatthe environmental conditions (nutrient availability,drought, etc.) met in the field are generally more severethan those in our greenhouse experiments. Therefore,we believe that the effects of hemiparasites, host plants,and their common herbivores on each other’s successmight be even more substantial in the field.

Our results indicate that the direct effects of para-sitism do not differ between cyanogenic and acyano-genic plants. However, we detected an indirect positiveeffect of parasitism on acyanogenic plants in terms ofreduced leaf area consumed by a generalist herbivore.This parasite-mediated difference in the effects of her-bivory between acyanogenic and cyanogenic plantsmay be one of the many genetic and environmentalfactors that regulate the cyanogenic polymorphism inT. repens. In lower latitudes or altitudes where T. re-pens populations have higher frequencies of cyano-genic plants (see Daday 1954b, Pederson et al. 1996),root hemiparasitism can be relevant to herbivore–whiteclover interactions by decreasing the difference in thelevel of herbivore damage between cyanogenic andacyanogenic plants.

To the best of our knowledge, we showed for thefirst time that food–plant parasitism by a hemiparasitesignificantly decreased the performance of a generalistherbivore feeding on the host species. Parasitic plantsand mollusk herbivores are common components ofgrassland plant communities and their interactions arecommon. Thus, in addition to the effects of parasiticplants on the structure and dynamics of plant com-munities (Gibson and Watkinson 1992, Pennings andCallaway 1996, Davies et al. 1997) parasitic plants mayhave indirect effects on the population dynamics ofherbivores and on the structure of herbivore commu-nities.

ACKNOWLEDGMENTS

Dr. Ian Rhodes from the Welsh Plant Breeding Station inAberystwyth kindly supplied the Trifolium repens seeds. PetriLeinonen from Agricultural Research Station of Finland, Par-tala, supplied the nitrogen-fixing bacteria. Kimmo Syrjanenand Sami Markkanen helped us to find Arianta arbustorum.We are grateful to Tuula Granfors, Paivi Kuusela, MarkettaKyyro-Uusitalo, Teija Linfors, Sirkku Punnonen, Pirita Puus-tinen, Pasi Salmi, and Eija Asikainen for their help in thegreenhouse and in the lab. We owe our warmest thanks toConchita Alonso, Jocelyn Martel, Juha Tuomi, Veikko Sa-

lonen, and Anthony Joern for valuable discussions or com-ments on earlier versions of the manuscript. The Ph.D. stu-dents in the Section of Ecology (University of Turku) alsogave valuable comments on an earlier version of the manu-script. This study was funded by the Academy of Finland andE. J. Sariola Foundation (to S. P).

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