Effects of antibacterial agents on in vitro ovine ruminal biotransformation of the hepatotoxic pyrrolizidine alkaloid jacobine.

Ingestion of pyrrolizidine alkaloids, naturally occurring plant toxins, causes illness and death in a number of animal species. Senecio jacobaea pyrrolizidine alkaloids cause significant economic losses due to livestock poisoning, particularly in the Pacific Northwest. Some sheep are resistant to pyrrolizidine alkaloid poisoning, because ovine ruminal biotransformation detoxifies free pyrrolizidine alkaloids in digesta. Antibacterial agents modify ruminal fermentation. Pretreatment with antibacterial agents may account for some animal variability in resistance to pyrrolizidine alkaloid toxicosis, and antibacterial agents can also be used for characterizing ruminal pyrrolizidine alkaloid-biotransforming microflora. The objective of this study was to evaluate the effects of antibacterial agents on biotransformation of a predominant S. jacobaea pyrrolizidine alkaloid, jacobine, in ovine ruminal contents. Ovine ruminal jacobine biotransformation was tested in vitro with 20 independent antibacterial agents. Low amounts of rifampin and erythromycin prevented jacobine biotransformation. Chlortetracycline, lasalocid, monensin, penicillin G, and tetracycline were slightly less effective at inhibiting jacobine biotransformation. Bacitracin, crystal violet, kanamycin, and neomycin were moderately inhibitory against jacobine biotransformation. Brilliant green, chloramphenicol, gramicidin, nalidixic acid, polymyxin B SO4, sodium azide, streptomycin, sulfisoxazole, and vancomycin had little to no effect on jacobine biotransformation. The antibiotics that were most effective at inhibiting biotransformation were those that are active against gram-positive bacteria. Therefore, gram-positive bacteria are most likely critical members of the jacobine-biotransforming consortia.     

Variation in pyrrolizidine alkaloid patterns of Senecio jacobaea

We studied the variation in pyrrolizidine alkaloid (PA) patterns of lab-grown vegetative plants of 11 European Senecio jacobaea populations. Plants were classified as jacobine, erucifoline, mixed or senecionine chemotypes based on presence and absence of the PAs jacobine or erucifoline. Due to the presence of jacobine, total PA concentration in jacobine chemotypes was higher than in erucifoline chemotypes. Both relative and absolute concentrations of individual PAs differed between half-sib and clonal families, which showed that variation in PA patterns had a genetic basis. Within most populations relative abundance of PAs varied considerably between individual plants. Most populations consisted either of the jacobine chemotype or of the erucifoline chemotype, sometimes in combination with mixed or senecionine chemotypes.     

Metabolism of toxic pyrrolizidine alkaloids from tansy ragwort (Senecio jacobaea) in ovine ruminal fluid under anaerobic conditions.

The ability of ovine ruminal fluid to metabolize pyrrolizidine alkaloid (PA) from Senecio jacobaea under anaerobic conditions was evaluated. Four fistulated sheep fed PA served as individual sources of ruminal fluid, which was incubated in a defined minimal salts medium under two different anaerobic conditions, denitrifying and methanogenic. Anaerobic cultures amended with ovine ruminal fluids (20%), PA (100 micrograms/ml), and a defined minimal salts medium were monitored for a period of several days. These cultures revealed that while PA was not depleted in sterile, autoclaved controls or under denitrifying conditions, it was metabolized during periods of active methanogenesis under methanogenic conditions. In addition, samples of ruminal fluid were separated by differential centrifugation under anaerobic conditions, and the resultant supernatants were tested for their ability to metabolize PA as compared with those of the respective uncentrifuged control fluids. Uncentrifuged controls exhibited a PA depletion rate of -4.04 +/- 0.17 micrograms of PA per ml per h. Supernatants 1 (centrifuged at 41 x g for 2 min), 2 (centrifuged at 166 x g for 5 min), and 3 (centrifuged at 1,500 x g for 10 min) exhibited significantly slower depletion rates, with slopes of data representing -1.64 +/- 0.16, -1.44 +/- 0.16, and -1.48 +/- 0.16 micrograms of PA metabolized per ml per h, respectively, demonstrating no statistically significant difference among the supernatant cultures. Microscopic evaluations revealed that protozoa were present in the control whole ruminal fluid and to a lesser extent in supernatant 1, while supernatants 2 and 3 contained only bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of toxic pyrrolizidine alkaloids from tansy ragwort (Senecio jacobaea) in ovine ruminal fluid under anaerobic conditions.

The ability of ovine ruminal fluid to metabolize pyrrolizidine alkaloid (PA) from Senecio jacobaea under anaerobic conditions was evaluated. Four fistulated sheep fed PA served as individual sources of ruminal fluid, which was incubated in a defined minimal salts medium under two different anaerobic conditions, denitrifying and methanogenic. Anaerobic cultures amended with ovine ruminal fluids (20%), PA (100 micrograms/ml), and a defined minimal salts medium were monitored for a period of several days. These cultures revealed that while PA was not depleted in sterile, autoclaved controls or under denitrifying conditions, it was metabolized during periods of active methanogenesis under methanogenic conditions. In addition, samples of ruminal fluid were separated by differential centrifugation under anaerobic conditions, and the resultant supernatants were tested for their ability to metabolize PA as compared with those of the respective uncentrifuged control fluids. Uncentrifuged controls exhibited a PA depletion rate of -4.04 +/- 0.17 micrograms of PA per ml per h. Supernatants 1 (centrifuged at 41 x g for 2 min), 2 (centrifuged at 166 x g for 5 min), and 3 (centrifuged at 1,500 x g for 10 min) exhibited significantly slower depletion rates, with slopes of data representing -1.64 +/- 0.16, -1.44 +/- 0.16, and -1.48 +/- 0.16 micrograms of PA metabolized per ml per h, respectively, demonstrating no statistically significant difference among the supernatant cultures. Microscopic evaluations revealed that protozoa were present in the control whole ruminal fluid and to a lesser extent in supernatant 1, while supernatants 2 and 3 contained only bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of endogenous avian tumor virus gene expression by pyrrolizidine alkaloids

The pyrrolizidine alkaloids (PA) are toxic compounds which occur naturally in plant species throughout the world. They have been implicated as both carcinogenic and mutagenic agents. An active metabolite of the alkaloids, the pyrrole, which is a strong alkylating agent, is thought to be the toxicant. The naturally occurring alkaloid, jacobine , is able to induce the production of endogenous avian RNA tumor virus particles in cultured chick embryo fibroblasts (CEF). When jacobine was modified to form retronecine it no longer induced virus particles. Conversion of retronecine to its pyrrole resulted in a compound capable of inducing virus particle production. The isobutyryl monoester of retronecine was also able to induce virus particle production, but the isobutyryl monoester pyrrole was unexpectedly inactive as an inducer. This type of viral induction system is useful for studying the effect of modification of the inducer on its biological activity.     

Host-plant choice and larval growth in the cinnabar moth: do pyrrolizidine alkaloids play a role?

Witte et al. (1992) described two distinct chemotypes of Senecio jacobaea L. Asteraceae, a chemotype with jacobine as one of the major pyrrolizidine alkaloids (PAs) and a chemotype with erucifoline as one of the major PAs. We hypothesized that the presence of erucifoline might be the factor responsible for the lack of success of the cinnabar moth on Senecio erucifolius L. Asteraceae and the S. jacobaea erucifoline chemotype. We performed a survey of the distribution of the two chemotypes in the Netherlands and compared this with the distribution map of Tyria jacobaeae L. Lepidoptera, Arctiidae. The distribution of the two chemotypes in the Netherlands is poorly correlated with the distribution of the cinnabar moth. The jacobine chemotype occurs along the coast and the erucifoline chemotype predominantly inward.An oviposition experiment showed that the cinnabar moth did not discriminate between the two chemotypes of S. jacobaea and S. erucifolius. Larval performance did not differ between the two chemotypes and species. Although the distribution of S. jacobaea jacobine chemotype is loosely associated with the abundance of the cinnabar moth the oviposition and growth experiments indicate that other factors than the presence of erucifoline play a role in this association.The absence of recordings of S. erucifolius as a foodplant for the cinnabar moth might be explained by the phenology of the foodplant. Ovipositing females of the univoltine cinnabar moth prefer flowering plants for oviposition. S. erucifolius starts flowering about 1–2 month later than S. jacobaea just after the peak density of moths.     

Testing chemical defence based on pyrrolizidine alkaloids

Pyrrolizidine alkaloids are considered the primary defence mechanism in aposematic ithomiine butterflies and arctiid moths. Despite evidence that pyrrolizidine alkaloids are effective against some invertebrate predators, proof for a protective function of pyrrolizidine alkaloids against vertebrate predators is fragmented. The present work shows that the pyrrolizidine alkaloid monocrot-aline is unpalatable to the pileated finch,Coryphospingus pileatusand that the unpalatability is learned through association with a specific colour pattern (blue stripes). In a series of trials, using mealworms as model prey, birds rejected those to which pyrrolizidine alkaloid solution had been applied topically but accepted prey devoid of the alkaloid. Subsequent offerings of prey with pyrrolizidine alkaloid and a painted blue-striped pattern led to consistent rejections by the experimental birds. Birds were then offered blue-striped painted larvae without pyrrolizidine alkaloids (‘mimics’), which were rejected at levels similar to the previous trial. The predators learned to recognize the prey as unpalatable items based on their experience in the previous encounters. These results provide evidence for the protective capacity of the pyrrolizidine alkaloid against a vertebrate predator and supports the role of these chemicals in aposematism in the Lepidoptera.     

Pyrrolizidine alkaloids in arctiid moths (Lep.) with a discussion on host plant relationships and the role of these secondary plant substances in the Arctiidae

Pyrrolizidine alkaloids (PA) have been identified in six species of Arctiidae reared on Senecio and Crotalaria. These include senecionine, seneciphylline, integerrimine, jacobine, jacozine, jacoline, jaconine and a metabolite (C₁₅H₂₅NO₅) from Senecio , and monocrotaline, trichodesmine and crispatine from Crotalaria.
The all-red aberration of Tyria jacobaeae (var. conyi) contained much less of the metabolite than normal examples of this species. Female Spilosoma lutea reared on the same plants of S. jacobaea contained markedly more jacobine and jacoline than die males.
Host plant relationships and secondary plant substances are discussed. It is suggested that the Arctiid moths' own deterrent secretions, directed against vertebrate predators, pre-adapts them for feeding on foliage likewise protected against large herbivores by toxic secondary plant substances such as cardenolides and pyrrolizidine alkaloids. These latter substances are more toxic to vertebrate than to insect herbivores, and their dual function of deterrent and insect aphrodisiac puts a premium on their sequestration and storage once a species has achieved the initial steps, and occupied the plant niche concerned. It is further suggested that the polyphagous habits of the Arctiidae result in a more equitable distribution of die secondary plant substances within the Mullerian complex concerned, thus providing a generalized warning message for the potential vertebrate predator.     

Molecular analysis of a consortium of ruminal microbes that detoxify pyrrolizidine alkaloids

Members of a consortium of bacteria, isolated from the rumen of sheep, that degrades pyrrolizidine alkaloids (PAs) found in tansy ragwort (Senecio jacobaea) were characterized. An enrichment of ruminal bacteria was isolated from a sample of ruminal fluid using standard anaerobic techniques. The PA degradative capacity of the enrichment was tested by spiking purified PA extract from tansy ragwort. Length heterogeneity analysis by PCR (LH-PCR) and restriction fragment length polymorphism (RFLP) analysis was used to identify members of the consortium. Phylogenetic analysis of the 16S rDNA gene revealed differing results based on the molecular method used. LH-PCR identified 7 different organisms in 3 groups while RFLP identified 6 organisms with differing banding patterns in 5 groups. After the phylogenetic analyses of both methods were combined, the combined isolates represented 6 groups. The majority of the members of this consortium are <97.0% homologous with known bacteria, indicating this consortium may contain novel organisms able to detoxify PAs found in tansy ragwort. Further understanding of the metabolic pathways used by this consortium to degrade PAs could lead to the use of the consortium as a probiotic therapy for livestock and horses afflicted with tansy ragwort toxicosis.     

The evolution of pyrrolizidine alkaloid diversity among and within Jacobaea species

Plants produce many secondary metabolites showing considerable inter- and intraspecific diversity of concentration and composition as a strategy to cope with environmental stresses. The evolution of plant defenses against herbivores and pathogens can be unraveled by understanding the mechanisms underlying chemical diversity. Pyrrolizidine alkaloids are a class of secondary metabolites with high diversity. We performed a qualitative and quantitative analysis of 80 pyrrolizidine alkaloids with liquid chromatography-tandem mass spectrometry of leaves from 17 Jacobaea species including one to three populations per species with 4–10 individuals per population grown under controlled conditions in a climate chamber. We observed large inter- and intraspecific variation in pyrrolizidine alkaloid concentration and composition, which were both species-specific. Furthermore, we sequenced 11 plastid and three nuclear regions to reconstruct the phylogeny of the 17 Jacobaea species. Ancestral state reconstruction at the species level showed mainly random distributions of individual pyrrolizidine alkaloids. We found little evidence for phylogenetic signals, as nine out of 80 pyrrolizidine alkaloids showed a significant phylogenetic signal for Pagel's λ statistics only, whereas no significance was detected for Blomberg's K measure. We speculate that this high pyrrolizidine alkaloid diversity is the result of the upregulation and downregulation of specific pyrrolizidine alkaloids depending on ecological needs rather than gains and losses of particular pyrrolizidine alkaloid biosynthesis genes during evolution.     

Independent recruitment of a flavin-dependent monooxygenase for safe accumulation of sequestered pyrrolizidine alkaloids in grasshoppers and moths

Several insect lineages have developed diverse strategies to sequester toxic pyrrolizidine alkaloids from food-plants for their own defense. Here, we show that in two highly divergent insect taxa, the hemimetabolous grasshoppers and the holometabolous butterflies, an almost identical strategy evolved independently for safe accumulation of pyrrolizidine alkaloids. This strategy involves a pyrrolizidine alkaloid N-oxygenase that transfers the pyrrolizidine alkaloids to their respective N-oxide, enabling the insects to avoid high concentrations of toxic pyrrolizidine alkaloids in the hemolymph. We have identified a pyrrolizidine alkaloid N-oxygenase, which is a flavin-dependent monooxygenase, of the grasshopper Zonocerus variegatus. After heterologous expression in E. coli, this enzyme shows high specificity for pyrrolizidine alkaloids of various structural types and for the tropane alkaloid atropine as substrates, a property that has been described previously for a pyrrolizidine alkaloid N-oxygenase of the arctiid moth Grammia geneura. Phylogenetic analyses of insect flavin-dependent monooxygenase sequences suggest that independent gene duplication events preceded the establishment of this specific enzyme in the lineages of the grasshoppers and of arctiid moths. Two further flavin-dependent monooxygenase sequences have been identified from Z. variegatus sharing amino acid identities of approximately 78% to the pyrrolizidine alkaloid N-oxygenase. After heterologous expression, both enzymes are also able to catalyze the N-oxygenation of pyrrolizidine alkaloids, albeit with a 400-fold lower specific activity. With respect to the high sequence identity between the three Z. variegatus sequences this ability to N-oxygenize pyrrolizidine alkaloids is interpreted as a relict of a former bifunctional ancestor gene of which one of the gene copies optimized this activity for the specific adaptation to pyrrolizidine alkaloid containing food plants.     

Pyrrolizidine alkaloid profiles of the Senecio cineraria group (Asteraceae)

Alkaloid profiles of five Senecio species (Asteraceae), including S. ambiguus subsp. ambiguus, S. ambiguus subsp. nebrodensis, S. gibbosus subsp. bicolor, S. gibbosus subsp. gibbosus, and S. gibbosus subsp. cineraria, were studied. Eleven pyrrolizidine alkaloids were identified and their content was evaluated by GLC-MS and GLC analysis. Otosenine and florosenine were found to be the major alkaloids in all studied species. It is interesting that only S. ambiguus subsp. nebrodensis was characterized by a high content of the alkaloids jacobine, jacoline, jaconine, and jacozine.     

Changes in Senecio grisebachii pyrrolizidine alkaloids abundances and profiles as response to soil quality

Abstract

Senecio grisebachii Baker is an invasive weed considered to be toxic due to the presence of pyrrolizidine alkaloids (PA) in its tissues. The PA production by S. grisebachii aerial parts was evaluated in samples grown in two Argentinean Rolling Pampa fields with the same kind of soil but differing in the length of their exploitation period by conventional tillage practices and, consequently, in their deterioration level.

We found significant differences in the relative concentrations of seven alkaloids between samples taken from the two fields. Seneciphylline was the most abundant alkaloid in inflorescences from less deteriorated soil (LD) while senecionine was the major one in those from highly deteriorated soil (D) being followed by seneciphylline, integerrimine, and minor amounts of spartiodine, jacobine, jacozine and retrorsine. A significant increase in total alkaloid content (TAC) was observed in inflorescences from samples growing in D soil (3.52±0.20 mg/g DW) when comparing with those from samples grown in LD one (3.23±0.26).     

The evolution of pyrrolizidine alkaloid biosynthesis and diversity in the Senecioneae

Pyrrolizidine alkaloids are characteristic secondary metabolites of the Asteraceae and some other plant families. They are especially numerous and diverse in the tribe Senecioneae and form a powerful defense mechanism against herbivores. Studies into the evolution of pyrrolizidine alkaloid biosynthesis using Senecio species have identified homospermidine synthase as the enzyme responsible for the synthesis of the first specific intermediate. These studies further indicated that the homospermidine synthase-encoding gene was recruited following gene duplication of deoxyhypusine synthase and that this occurred independently in several different angiosperm lineages. A review of published pyrrolizidine alkaloid data shows that the Senecioneae are characterized by a large qualitative and quantitative variation in pyrrolizidine alkaloid profiles and that these data demonstrate little phylogenetic signal. This suggests that although the first steps of this pathway are highly conserved, the diversification of secondarily derived pyrrolizidine alkaloids is extremely plastic.     

Concomitant occurrence of pyrrolizidine and quinolizidine alkaloids in the hemiparasite Osyris alba L. (Santalaceae)

The investigation of the alkaloid extracts of the hemiparasitic plant Osyris alba, collected from three different localities in southern France, revealed the concomitant presence of both pyrrolizidine (PA) and quinolizidine (QA) alkaloids in the samples from two of these localities. The sample from the third locality contained only PAs. The eight QAs identified were sparteine, N-methylcytisine, cytisine, methyl-12-cytisine acetate, hydroxy-N-methylcytisine, N-acetylcytisine, lupanine, and anagyrine. Of the eleven detected PAs, eight were identified as chysin A, chysin B, 1-carboxypyrrolizidine-7-olide, senecionine, integerrimine, retrorsine, senecivernine and a new alkaloid janfestine (7R-hydroxychysin A or 1R-carbomethoxy-7R-hydroxypyrrolizidine). PAs were mainly present as their N-oxides This is, to our knowledge, the first report demonstrating the simultaneous presence of two classes of alkaloids, quinolizidine and pyrrolizidine alkaloids, in a single parasitic plant. As these alkaloids do not occur in the same host plant, the results indicate that Osyris must have tapped more than one host plant concomitantly. Since both quinolizidine and pyrrolizidine alkaloids serve as defence compounds against herbivores, affecting different molecular targets, the simultaneous acquisition of the two types of alkaloids by a single plant could provide a novel mode of defence of hemiparasites against herbivores.     

Pyrrolizidine alkaloids in and on the leaf surface of Senecio jacobaea L

This is the first study showing that alkaloids are present on the leaf surface of plants. A concentration of 30-230 pmol/cm2 pyrrolizidine alkaloids (PA's) was detected in 8 different samples taken from Senecio jacobaea. PA concentration on the leaves was marginally correlated with PA concentration of the total leaf tissues. The PA spectrum on the leaf differed from the PA spectrum of the total leaf.     

Biochemical strategy of sequestration of pyrrolizidine alkaloids by adults and larvae of chrysomelid leaf beetles

Tracer feeding experiments with (14)C-labeled senecionine and senecionine N-oxide were carried out to identify the biochemical mechanisms of pyrrolizidine alkaloid sequestration in the alkaloid-adapted leaf beetle Oreina cacaliae (Chrysomelidae). The taxonomically closely related mint beetle (Chrysolina coerulans) which in its life history never faces pyrrolizidine alkaloids was chosen as a 'biochemically naive' control. In C. coerulans ingestion of the two tracers resulted in a transient occurrence of low levels of radioactivity in the hemolymph (1-5% of radioactivity fed). With both tracers, up to 90% of the radioactivity recovered from the hemolymph was senecionine. This indicates reduction of the alkaloid N-oxide in the gut. Adults and larvae of O. cacaliae sequester ingested senecionine N-oxide almost unchanged in their bodies (up to 95% of sequestered total radioactivity), whereas the tertiary alkaloid is converted into a polar metabolite (up to 90% of total sequestered radioactivity). This polar metabolite, which accumulates in the hemolymph and body, was identified by LC/MS analysis as an alkaloid glycoside, most likely senecionine O-glucoside. The following mechanism of alkaloid sequestration in O. cacaliae is suggested to have developed during the evolutionary adaptation of O. cacaliae to its alkaloid containing host plant: (i) suppression of the gut specific reduction of the alkaloid N-oxides, (ii) efficient uptake of the alkaloid N-oxides, and (iii) detoxification of the tertiary alkaloids by O-glucosylation. The biochemical mechanisms of sequestration of pyrrolizidine alkaloid N-oxides in Chysomelidae leaf beetles and Lepidoptera are compared with respect to toxicity, safe storage and defensive role of the alkaloids.     

Tissue distribution and biosynthesis of 1,2-saturated pyrrolizidine alkaloids in Phalaenopsis hybrids (Orchidaceae)

Phalaenopsis hybrids contain two 1,2-saturated pyrrolizidine monoesters, T-phalaenopsine (necine base trachelanthamidine) and its stereoisomer Is-phalaenopsine (necine base isoretronecanol). T-Phalaenopsine is the major alkaloid accounting for more than 90% of total alkaloid. About equal amounts of alkaloid were genuinely present as free base and its N-oxide. The structures were confirmed by GC-MS. The quantitative distribution of phalaenopsine in various organs and tissues of vegetative rosette plants and flowering plants revealed alkaloid in all tissues. The highest concentrations were found in young and developing tissues (e.g., root tips and young leaves), peripheral tissues (e.g., of flower stalks) and reproductive organs (flower buds and flowers). Within flowers, parts that usually attract insect visitors (e.g., labellum with colorful crests as well as column and pollinia) show the highest alkaloid levels. Tracer feeding experiments with (14)C-labeled putrecine revealed that in rosette plants the aerial roots were the sites of phalaenopsine biosynthesis. However active biosynthesis was only observed in roots still attached to the plant but not in excised roots. There is a slow but substantial translocation of newly synthesized alkaloid from the roots to other plant organs. A long-term tracer experiment revealed that phalaenopsine shows neither turnover nor degradation. The results are discussed in the context of a polyphyletic molecular origin of the biosynthetic pathways of pyrrolizidine alkaloids in various scattered angiosperm taxa. The ecological role of the so called non-toxic 1,2-saturated pyrrolizidine alkaloids is discussed in comparison to the pro-toxic 1,2-unsaturated pyrrolizidine alkaloids. Evidence from the plant-insect interphase is presented indicating a substantial role of the 1,2-saturated alkaloids in plant and insect defense.     

The mutagenic activity of some pyrrolizidine alkaloids inDrosophila

Summary Nine pyrrolizidine alkaloids have been tested by the injection method for their ability to produce sex-linked recessive lethals in adult males ofDrosophila melanogaster. At a concentration of 0.02 M, monocrotaline, lasiocarpine and heliotrine were found to be strongly mutagenic. Echinatine, echimidine, senecionine and supinine were less active, while jacobine and platyphylline were only feebly mutagenic. In four cases tested, the N-oxides were found to be less active than their parent bases. The results are discussed in relation to the possibility that some of these alkaloids may be carcinogenic in man.With 1 Figure in the Text     

Chemotype of <Emphasis Type="Italic">Senecio jacobaea</Emphasis> affects damage by pathogens and insect herbivores in the field

The evolution of the diversity of plant secondary metabolites is still poorly understood. To determine whether natural enemies could exert selection on plant secondary chemistry, pathogen infestation and invertebrate herbivory were measured on 10 genotypes of Senecio jacobaea (Tansy Ragwort) at two experimental field sites during a 2-year period. The genotypes represented two chemotypes based on the presence of the pyrrolizidine alkaloids (PA) jacobine and erucifoline. At one site, Heteren, mainly generalist herbivores were present. Here, damage was limited and did not differ among genotypes or chemotypes. At the other site, Meijendel, several specialists attacked the plants. Damage increased during the year, with a peak in July when most damage was caused by the specialist moth Tyria jacobaeae. At this peak there was no difference in damage among chemotypes. In the months prior to T. jacobaeae damage, chemotypes with jacobine were more severely attacked by specialists than the chemotypes without jacobine. Total damage during that period was positively correlated with both total PA concentration and jacobine concentration. Probably plant vigor also played a role in host preference since damage per individual plant was positively correlated with plant size. Our results suggest that total PA concentration and specifically jacobine had a positive effect on specialist feeding, indicating ecological costs involved in the production of PAs. Ecological costs related to plant secondary compounds could explain why not all individuals produce high levels of these compounds. In addition, differences in specialist herbivore pressures among sites may contribute to the variation in secondary metabolites among populations.