Bioassay-directed analysis-based identification of relevant pyrrolizidine alkaloids.

Pyrrolizidine alkaloids (PAs) are produced by various plant species and have been detected as contaminants in food and feed. Monitoring programmes should include PAs that are present in relevant matrices and that exhibit a high toxic potential. The aim of the present study was to use a bioassay-directed analysis approach to identify relevant PAs not yet included in monitoring programmes. To that end, extracts of Heliotropium europaeum and H. popovii were prepared and analysed with LC-MS/MS for the presence of 35 PAs included in monitoring programmes, as well as for genotoxic activity in the HepaRG/γH2AX assay. Europine, heliotrine and lasiocarpine were found to be the most abundant PAs. The extracts showed a higher γH2AX activity than related artificial mixtures of quantified known PAs, which might point to the presence of unknown toxic PAs. The H. europaeum extract was fractionated and γH2AX activities of individual fractions were determined. Fractions were further analysed applying LC-Orbitrap-MS analysis and Compound Discoverer software, identifying various candidate PAs responsible for the non-explained genotoxic activity. Altogether, the results obtained show that bioassay-directed analysis allows identification of candidate PAs that can be included in monitoring programmes.

Novel Insights into Pyrrolizidine Alkaloid Toxicity and Implications for Risk Assessment: Occurrence, Genotoxicity, Toxicokinetics, Risk Assessment-A Workshop Report.

This paper reports on the major contributions and results of the 2nd International Workshop of Pyrrolizidine Alkaloids held in September 2020 in Kaiserslautern, Germany. Pyrrolizidine alkaloids are among the most relevant plant toxins contaminating food, feed, and medicinal products of plant origin. Hundreds of PA congeners with widespread occurrence are known, and thousands of plants are assumed to contain PAs. Due to certain PAs' pronounced liver toxicity and carcinogenicity, their occurrence in food, feed, and phytomedicines has raised serious human health concerns. This is particularly true for herbal teas, certain food supplements, honey, and certain phytomedicinal drugs. Due to the limited availability of animal data, broader use of in vitro data appears warranted to improve the risk assessment of a large number of relevant, 1,2-unsaturated PAs. This is true, for example, for the derivation of both toxicokinetic and toxicodynamic data. These efforts aim to understand better the modes of action, uptake, metabolism, elimination, toxicity, and genotoxicity of PAs to enable a detailed dose-response analysis and ultimately quantify differing toxic potencies between relevant PAs. Accordingly, risk-limiting measures comprising production, marketing, and regulation of food, feed, and medicinal products are discussed.

Comparative Risk Assessment of Three Native Heliotropium Species in Israel.

Pyrrolizidine alkaloids (PAs) are genotoxic carcinogenic phytotoxins mostly prevalent in the Boraginaceae, Asteraceae and Fabaceae families. Heliotropium species (Boraginaceae) are PA-producing weeds, widely distributed in the Mediterranean region, that have been implicated with lethal intoxications in livestock and humans. In Israel, H. europaeum, H. rotundifolium and H. suaveolens are the most prevalent species. The toxicity of PA-producing plants depends on the PA concentration and composition. PAs occur in plants as mixtures of dozens of various PA congeners. Hence, the risk arising from simultaneous exposure to different congeners has to be evaluated. The comparative risk evaluation of the three Heliotropium species was based on recently proposed interim relative potency (iREP) factors, which take into account certain structural features as well as in vitro and in vivo toxicity data obtained for several PAs of different classes. The aim of the present study was to determine the PA profile of the major organ parts of H. europaeum, H. rotundifolium and H. suaveolens in order to assess the plants' relative toxic potential by utilizing the iREP concept. In total, 31 different PAs were found, among which 20 PAs were described for the first time for H. rotundifolium and H. suaveolens. The most prominent PAs were heliotrine-N-oxide, europine-N-oxide and lasiocarpine-N-oxide. Europine-N-oxide displayed significant differences among the three species. The PA levels ranged between 0.5 and 5% of the dry weight. The flowers of the three species were rich in PAs, while the PA content in the root and flowers of H. europaeum was higher than that of the other species. H. europaeum was found to pose a higher risk to mammals than H. rotundifolium, whereas no differences were found between H. europaeum and H. suaveolens as well as H. suaveolens and H. rotundifolium.

Removal of soil biota alters soil feedback effects on plant growth and defense chemistry.

We examined how the removal of soil biota affects plant-soil feedback (PSF) and defense chemistry of Jacobaea vulgaris, an outbreak plant species in Europe containing the defense compounds pyrrolizidine alkaloids (PAs). Macrofauna and mesofauna, as well as fungi and bacteria, were removed size selectively from unplanted soil or soil planted with J. vulgaris exposed or not to above- or belowground insect herbivores. Wet-sieved fractions, using 1000-, 20-, 5- and 0.2-µm mesh sizes, were added to sterilized soil and new plants were grown. Sieving treatments were verified by molecular analysis of the inocula. In the feedback phase, plant biomass was lowest in soils with 1000- and 20-µm inocula, and soils conditioned with plants gave more negative feedback than without plants. Remarkably, part of this negative PSF effect remained present in the 0.2-µm inoculum where no bacteria were present. PA concentration and composition of plants with 1000- or 20-µm inocula differed from those with 5- or 0.2-µm inocula, but only if soils had been conditioned by undamaged plants or plants damaged by aboveground herbivores. These effects correlated with leaf hyperspectral reflectance. We conclude that size-selective removal of soil biota altered PSFs, but that these PSFs were also influenced by herbivory during the conditioning phase.

Methyl Jasmonate Changes the Composition and Distribution Rather than the Concentration of Defence Compounds: a Study on Pyrrolizidine Alkaloids.

In this study we investigated the effect of methyl jasmonate (MeJA) application on pyrrolizidine alkaloid (PA) concentration and composition of two closely related Jacobaea species. In addition, we examined whether MeJA application affected herbivory of the polyphagous leaf feeding herbivore Spodoptera exigua. A range of concentrations of MeJA was added to the medium of Jacobaea vulgaris and J. aquatica tissue culture plants grown under axenic conditions. PA concentrations were measured in roots and shoots using LC-MS/MS. In neither species MeJA application did affect the total PA concentration at the whole plant level. In J. vulgaris the total PA concentration decreased in roots but increased in shoots. In J. aquatica a similar non-significant trend was observed. In both Jacobaea species MeJA application induced a strong shift from senecionine- to erucifoline-like PAs, while the jacobine- and otosenine-like PAs remained largely unaffected. The results show that MeJA application does not necessarily elicits de novo synthesis, but rather leads to PA conversion combined with reallocation of certain PAs from roots to shoots. S. exigua preferred feeding on control leaves of J. aquatica over MeJA treated leaves, while for J. vulgaris both the control and MeJA treated leaves were hardly eaten. This suggests that the MeJA-induced increase of erucifoline-like PAs can play a role in resistance of J. aquatica to S. exigua. In J. vulgaris resistance to S. exigua may already be high due to the presence of jacobine-like PAs or other resistance factors.

Risk assessment for pyrrolizidine alkaloids detected in (herbal) teas and plant food supplements.

Pyrrolizidine alkaloids (PAs) are plant metabolites present in some botanical preparations, with especially 1,2-unsaturated PAs being of concern because they are genotoxic carcinogens. This study presents an overview of tumour data on PAs and points of departure (PODs) derived from them, corroborating that the BMDL10 for lasiocarpine represents a conservative POD for risk assessment. A risk assessment using this BMDL10 and mean levels of PAs reported in literature for (herbal) teas, indicates that consumption of one cup of tea a day would result in MOE values lower than 10 000 for several types of (herbal) teas, indicating a priority for risk management for these products A refined risk assessment using interim relative potency (REP) factors showed that based on the mean PA levels, 7(54%) of 13 types of (herbal) teas and 1 (14%) of 7 types of plant food supplements (PFS) resulted in MOE values lower than 10 000, indicating a priority for risk management also for these products in particular. This includes both preparations containing PA-producing and non-PA-producing plants. Our study provides insight in the current state-of-the art and limitations in the risk assessment of PA-containing food products, especially (herbal) teas and PFS, indicating that PAs in food presents a field of interest for current and future risk management.

Disentangling above- and belowground neighbor effects on the growth, chemistry, and arthropod community on a focal plant.

Neighboring plants can influence arthropods on a focal plant, and this can result in associational resistance or associational susceptibility. These effects can be mediated by above- and belowground interactions between the neighbor and focal plant, but determining the relative contribution of the above- and belowground effects remains an open challenge. We performed a common garden experiment with a design that enabled us to disentangle the above- and belowground effects of five different plant species on the growth and chemistry of the focal plant ragwort (Jacobaea vulgaris), and the arthropod community associated with this plant. Aboveground effects of different neighboring plant species were more important for the growth and quality of J. vulgaris and for the arthropod abundance on this plant than belowground effects of neighbors. This remained true when only indirect neighbor effects (via affecting the biomass or quality of the focal plant) were considered. The aboveground neighbor effects on arthropod abundance on the focal plant were strongly negative. However, the magnitude of the effect depended on the identity of the neighboring species, and herbivore abundance on the focal plant was higher when surrounded by conspecific than when surrounded by heterospecific plants. We also observed interactions between above- and belowground neighbor effects, indicating that these effects may be nonadditive. We conclude that above- and belowground associational effects are not equally strong, and that neighbor effects on plant-arthropod interactions occur predominantly aboveground.

Testing the generalist-specialist dilemma: the role of pyrrolizidine alkaloids in resistance to invertebrate herbivores in Jacobaea species.

Plants produce a diversity of secondary metabolites (SMs) to protect them from generalist herbivores. On the other hand, specialist herbivores use SMs for host plant recognition, feeding and oviposition cues, and even sequester SMs for their own defense. Therefore, plants are assumed to face an evolutionary dilemma stemming from the contrasting effects of generalist and specialist herbivores on SMs. To test this hypothesis, bioassays were performed with F2 hybrids from Jacobaea species segregating for their pyrrolizidine alkaloids (PAs), using a specialist flea beetle (Longitarsus jacobaeae) and a generalist slug (Deroceras invadens). Our study demonstrated that while slug feeding damage was negatively correlated with the concentration of total PAs and that of senecionine-like PAs, flea beetle feeding damage was not affected by PAs. It was positively correlated though, with leaf fresh weight. The generalist slug was deterred by senecionine-like PAs but the specialist flea beetle was adapted to PAs in its host plant. Testing other herbivores in the same plant system, it was observed that the egg number of the specialist cinnabar moth was positively correlated with jacobine-like PAs, while the silver damage of generalist thrips was negatively correlated with senecionine- and jacobine-like PAs, and the pupae number of generalist leaf miner was negatively correlated with otosenine-like PAs. Therefore, while the specialist herbivores showed no correlation whatsoever with PA concentration, the generalist herbivores all showed a negative correlation with at least one type of PA. We concluded that the generalist herbivores were deterred by different structural groups of PAs while the specialist herbivores were attracted or adapted to PAs in its host plants.

OsJAR1 is required for JA-regulated floret opening and anther dehiscence in rice.

Jasmonates are important phytohormones regulating reproductive development. We used two recessive rice Tos17 alleles of OsJAR1, osjar1-2 and osjar1-3, to study the biological function of jasmonates in rice anthesis. The florets of both osjar1 alleles stayed open during anthesis because the lodicules, which control flower opening in rice, were not withering on time. Furthermore, dehiscence of the anthers filled with viable pollen, was impaired, resulting in lower fertility. In situ hybridization and promoter GUS transgenic analysis confirmed OsJAR1 expression in these floral tissues. Flower opening induced by exogenous applied methyl jasmonate was impaired in osjar1 plants and was restored in a complementation experiment with transgenics expressing a wild type copy of OsJAR1 controlled by a rice actin promoter. Biochemical analysis showed that OsJAR1 encoded an enzyme conjugating jasmonic acid (JA) to at least Ile, Leu, Met, Phe, Trp and Val and both osjar1 alleles had substantial reduction in content of JA-Ile, JA-Leu and JA-Val in florets. We conclude that OsJAR1 is a JA-amino acid synthetase that is required for optimal flower opening and closing and anther dehiscence in rice.

Development and validation of a rapid multiplex ELISA for pyrrolizidine alkaloids and their N-oxides in honey and feed.

Pyrrolizidine alkaloids (PAs) are a group of plant secondary metabolites with carcinogenic and hepatotoxic properties. When PA-producing plants contaminate crops, toxins can be transferred through the food chain and cause illness in humans and animals, most notably hepatic veno-occlusive disease. Honey has been identified as a direct risk of human exposure. The European Food Safety Authority has recently identified four groups of PAs that are of particular importance for food and feed: senecionine-type, lycopsamine-type, heliotrine-type and monocrotaline-type. Liquid or gas chromatography methods are currently used to detect PAs but there are no rapid screening assays available commercially. Therefore, the aim of this study was to develop a rapid multiplex ELISA test for the representatives of three groups of alkaloids (senecionine, lycopsamine and heliotrine types) that would be used as a risk-management tool for the screening of these toxic compounds in food and feed. The method was validated for honey and feed matrices and was demonstrated to have a detection capability less than 25 µg/kg for jacobine, lycopsamine, heliotrine and senecionine. The zinc reduction step introduced to the extraction procedure allows for the additional detection of the presence of N-oxides of PAs. This first multiplex immunoassay for PA detection with N-oxide reduction can be used for the simultaneous screening of 21 samples for >12 PA analytes. Honey samples (n = 146) from various origins were analysed for PA determination. Six samples were determined to contain measurable PAs >25 µg/kg by ELISA which correlated to >10 µg/kg by LC-MS/MS.

New Analytical Approach to Quinolizidine Alkaloids and Their Assumed Biosynthesis Pathways in Lupin Seeds.

Alkaloids play an essential role in protecting plants against herbivores. Humans can also benefit from the pharmacological effects of these compounds. Plants produce an immense variety of structurally different alkaloids, including quinolizidine alkaloids, a group of bi-, tri-, and tetracyclic compounds produced by Lupinus species. Various lupin species produce different alkaloid profiles. To study the composition of quinolizidine alkaloids in lupin seeds, we collected 31 populations of two wild species native to Israel, L. pilosus and L. palaestinus, and analyzed their quinolizidine alkaloid contents. Our goal was to study the alkaloid profiles of these two wild species to better understand the challenges and prospective uses of wild lupins. We compared their profiles with those of other commercial and wild lupin species. To this end, a straightforward method for extracting alkaloids from seeds and determining the quinolizidine alkaloid profile by LC-MS/MS was developed and validated in-house. For the quantification of quinolizidine alkaloids, 15 analytical reference standards were used. We used GC-MS to verify and cross-reference the identity of certain alkaloids for which no analytical standards were available. The results enabled further exploration of quinolizidine alkaloid biosynthesis. We reviewed and re-analyzed the suggested quinolizidine alkaloid biosynthesis pathway, including the relationship between the amino acid precursor l-lysine and the different quinolizidine alkaloids occurring in seeds of lupin species. Revealing alkaloid compositions and highlighting some aspects of their formation pathway are important steps in evaluating the use of wild lupins as a novel legume crop.

Recent advances and challenges in the analysis of natural toxins.

Natural toxins (NTs) are poisonous secondary metabolites produced by living organisms developed to ward off predators. Especially low molecular weight NTs (MW<∼1 kDa), such as mycotoxins, phycotoxins, and plant toxins, are considered an important and growing food safety concern. Therefore, accurate risk assessment of food and feed for the presence of NTs is crucial. Currently, the analysis of NTs is predominantly performed with targeted high pressure liquid chromatography tandem mass spectrometry (HPLC-MS/MS) methods. Although these methods are highly sensitive and accurate, they are relatively expensive and time-consuming, while unknown or unexpected NTs will be missed. To overcome this, novel on-site screening methods and non-targeted HPLC high resolution mass spectrometry (HRMS) methods have been developed. On-site screening methods can give non-specialists the possibility for broad "scanning" of potential geographical regions of interest, while also providing sensitive and specific analysis at the point-of-need. Non-targeted chromatography-HRMS methods can detect unexpected as well as unknown NTs and their metabolites in a lab-based approach. The aim of this chapter is to provide an insight in the recent advances, challenges, and perspectives in the field of NTs analysis both from the on-site and the laboratory perspective.

Effects of dietary exposure to plant toxins on bioaccumulation, survival, and growth of black soldier fly (Hermetia illucens) larvae and lesser mealworm (Alphitobius diaperinus).

In their natural habitat, insects may bioaccumulate toxins from plants for defence against predators. When insects are accidently raised on feed that is contaminated with toxins from co-harvested herbs, this may pose a health risk when used for human or animal consumption. Plant toxins of particular relevance are the pyrrolizidine alkaloids (PAs), which are genotoxic carcinogens produced by a wide variety of plant species and the tropane alkaloids (TAs) which are produced by a number of Solanaceae species. This study aimed to investigate the transfer of these plant toxins from substrates to black soldier fly larvae (BSFL) and lesser mealworm (LMW). PAs and the TAs atropine and scopolamine were added to insect substrate simulating the presence of different PA- or TA-containing herbs, and BSFL and LMW were grown on these substrates. Bioaccumulation from substrate to insects varied widely among the different plant toxins. Highest bioaccumulation was observed for the PAs europine, rinderine and echinatine. For most PAs and for atropine and scopolamine, bioaccumulation was very low. In the substrate, PA N-oxides were quickly converted to the corresponding tertiary amines. More research is needed to verify the findings of this study at larger scale, and to determine the potential role of the insect and/or substrate microbiome in metabolizing these toxins.

Effects of root herbivory on pyrrolizidine alkaloid content and aboveground plant-herbivore-parasitoid interactions in Jacobaea vulgaris.

The importance of root herbivory is increasingly recognized in ecological studies, and the effects of root herbivory on plant growth, chemistry, and performance of aboveground herbivores have been relatively well studied. However, how belowground herbivory by root feeding insects affects aboveground parasitoid development is largely unknown. In this study, we examined the effects of root herbivory by wireworms (Agriotes lineatus) on the expression of primary and secondary compounds in the leaves and roots of ragwort (Jacobaea vulgaris). We also studied the effects of root herbivory on the performance of a generalist aboveground herbivore, Mamestra brassicae and its parasitoid Microplitis mediator. In contrast to what most other studies have reported, root herbivory in J. vulgaris had a strong negative effect on the total concentration of pyrrolizidine alkaloids (PAs) in shoot tissues. The composition of PAs in the shoots also changed after root herbivory. In particular, the concentration of less toxic N-oxide PAs decreased. There was no significant effect of root herbivory on PA composition and concentration in the roots. Although the concentration of PA in the leaves decreased, M. brassicae tended to grow slower on the plants exposed to root herbivory. Parasitoid performance was not affected by root herbivory, but parasitoids developed faster when the concentration of jacobine-type PAs in the foliage was higher. These results point at a putative role of individual PAs in multitrophic interactions and emphasize that generalizations about aboveground-belowground effects should be made with great caution.

Pyrrolizidine alkaloid composition influences cinnabar moth oviposition preferences in Jacobaea hybrids.

Plants produce a variety of secondary metabolites (PSMs) that may be selective against herbivores. Yet, specialist herbivores may use PSMs as cues for host recognition, oviposition, and feeding stimulation, or for their own defense against parasites and predators. This summarizes a dual role of PSMs: deter generalists but attract specialists. It is not clear yet whether specialist herbivores are a selective force in the evolution of PSM diversity. A prerequisite for such a selective force would be that the preference and/or performance of specialists is influenced by PSMs. To investigate these questions, we conducted an oviposition experiment with cinnabar moths (Tyria jacobaeae) and plants from an artificial hybrid family of Jacobaea vulgaris and Jacobaea aquatica. The cinnabar moth is a specialist herbivore of J. vulgaris and is adapted to pyrrolizidine alkaloids (PAs), defensive PSMs of these plants. The number of eggs and egg batches oviposited by the moths were dependent on plant genotype and positively correlated to concentrations of tertiary amines of jacobine-like PAs and some otosenine-like PAs. The other PAs did not correlate with oviposition preference. Results suggest that host plant PAs influence cinnabar moth oviposition preference, and that this insect is a potential selective factor against a high concentration of some individual PAs, especially those that are also involved in resistance against generalist herbivores.

Emerging Issues on Tropane Alkaloid Contamination of Food in Europe.

The occurrence of tropane alkaloids (TAs), toxic plant metabolites, in food in Europe was studied to identify those TAs in food most relevant for human health. Information was extracted from the literature and the 2016 study from the European Food Safety Authority. Calystegines were identified as being inherent TAs in foods common in Europe, such as Solanum tuberosum (potato), S. melongena (eggplant, aubergine), Capsicum annuum (bell pepper) and Brassica oleracea (broccoli, Brussels sprouts). In addition, some low-molecular-weight tropanes and Convolvulaceae-type TAs were found inherent to bell pepper. On the other hand, atropine, scopolamine, convolvine, pseudotropine and tropine were identified as emerging TAs resulting from the presence of associated weeds in food. The most relevant food products in this respect are unprocessed and processed cereal-based foods for infants, young children or adults, dry (herbal) teas and canned or frozen vegetables. Overall, the occurrence data on both inherent as well as on associated TAs in foods are still scarce, highlighting the need for monitoring data. It also indicates the urge for food safety authorities to work with farmers, plant breeders and food business operators to prevent the spreading of invasive weeds and to increase awareness.

Occurrence and associated health risks of pyrrolizidine alkaloids in supplements marketed in Ghana for improved sexual performance.

Pyrrolizidine alkaloids (PAs) are noted for their hepatotoxic, genotoxic, and carcinogenic effects in animals and humans following metabolic activation in the liver. In this study, herbal supplements sold in Ghana for sexual improvement were analysed for the presence of 64 PAs using LC-MS/MS analysis. Up to 17 different PAs were identified in 19 out of the 37 samples analysed. The sum of PAs in samples ranged from 5 to 3204 µg kg-1. Since the PA content in the herbal medicinal preparations was generally lower than in honey samples, their presence was mainly attributed to cross-contamination. The observed levels would result in estimated daily intakes from 0.01 to 12 µg per day or 0.0002 to 0.2 µg kg-1 bw day-1 for a person weighing 70 kg. The margins of exposure ranged from 1200 to 1,400,000 with eight samples showing values below 10,000, thus indicating a health concern.

Legacy effects of aboveground-belowground interactions.

Root herbivory can greatly affect the performance of aboveground insects via changes in plant chemistry. These interactions have been studied extensively in experiments where aboveground and belowground insects were feeding on the same plant. However, little is known about how aboveground and belowground organisms interact when they feed on plant individuals that grow after each other in the same soil. We show that feeding by aboveground and belowground insect herbivores on ragwort (Jacobaea vulgaris) plants exert unique soil legacy effects, via herbivore-induced changes in the composition of soil fungi. These changes in the soil biota induced by aboveground and belowground herbivores of preceding plants greatly influenced the pyrrolizidine alkaloid content, biomass and aboveground multitrophic interactions of succeeding plants. We conclude that plant-mediated interactions between aboveground and belowground insects are also important when they do not feed simultaneously on the same plant.

Pyrrolizidine alkaloid variation in shoots and roots of segregating hybrids between Jacobaea vulgaris and Jacobaea aquatica.

Hybridization can lead to novel qualitative or quantitative variation of secondary metabolite (SM) expression that can have ecological and evolutionary consequences. We measured pyrrolizidine alkaloid (PA) expression in the shoots and roots of a family including one Jacobaea vulgaris genotype and one Jacobaea aquatica genotype (parental genotypes), two F(1) hybrid genotypes, and 102 F(2) hybrid genotypes using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We detected 37 PAs in the roots and shoots of J. vulgaris, J. aquatica and the hybrids. PA concentrations and compositions differed between genotypes, and between roots and shoots. Three otosenine-like PAs that only occurred in the shoots of parental genotypes were present in the roots of F(2) hybrids; PA compositions were sometimes novel in F(2) hybrids compared with parental genotypes, and in some cases transgressive PA expression occurred. We also found that PAs from within structural groups covaried both in the roots and in the shoots, and that PA expression was correlated between shoots and roots. Considerable and novel variation present among F(2) hybrids indicates that hybridization has a potential role in the evolution of PA diversity in the genus Jacobaea, and this hybrid system is useful for studying the genetic control of PA expression.

The relationship between structurally different pyrrolizidine alkaloids and western flower thrips resistance in F(2) hybrids of Jacobaea vulgaris and Jacobaea aquatica.

Segregating plant hybrids often have more ecological and molecular variability compared to parental species, and are therefore useful for studying relationships between different traits, and the adaptive significance of trait variation. Hybrid systems have been used to study the relationship between the expression of plant defense compounds and herbivore susceptibility. We conducted a western flower thrips (WFT) bioassay using a hybrid family and investigated the relationship between WFT resistance and pyrrolizidine alkaloid (PA) variation. The hybrid family consisted of two parental (Jacobaea vulgaris and Jacobaea aquatica) genotypes, two F(1) genotypes, and 94 F(2) hybrid lines. The J. aquatica genotype was more susceptible to thrips attack than the J. vulgaris genotype, the two F(1) hybrids were as susceptible as J. aquatica, and susceptibility to WFT differed among F(2) hybrid lines: 69 F(2) lines were equally susceptible compared to J. aquatica, 10 F(2) lines were more susceptible than J. aquatica and 15 F(2) lines were as resistant as J. vulgaris or were intermediate to the two parental genotypes. Among 37 individual PAs that were derived from four structural groups (senecionine-, jacobine-, erucifoline- and otosenine-like PAs), the N-oxides of jacobine, jaconine, and jacoline were negatively correlated with feeding damage caused by WFT, and the tertiary amines of jacobine, jaconine, jacoline, and other PAs did not relate to feeding damage. Total PA concentration was negatively correlated with feeding damage. Among the four PA groups, only the total concentration of the jacobine-like PAs was negatively correlated with feeding damage. Multiple regression tests suggested that jacobine-like PAs play a greater role in WFT resistance than PAs from other structural groups. We found no evidence for synergistic effects of different PAs on WFT resistance. The relationship between PA variation and WFT feeding damage in the Jacobaea hybrids suggests a role for PAs in resistance to generalist insects.